Part of The Super Mario Galaxy Movie. For characters, objects, and species, see Characters, objects and species.

Far out in the galaxy, Rosalina drifts through space aboard the Comet Observatory, raising a brood of Luma children as their mother. One evening, just as she settles in to read them a bedtime story, the observatory is set upon by Megaleg, whose pilot demands she surrender herself. Rosalina battles the machine and nearly tears it apart, but the fight stalls when a frightened Luma wanders out of the library seeking her. Spotting the children, the pilot turns Megaleg's fire on them, and Rosalina throws herself between the cannon and the library to shield them. She is ultimately captured, and the pilot reveals himself as Bowser Jr., Bowser's son, who intends to rebuild the family name with Kamek at his side. A Luma that tries to free her is seized as well, and the rest can only watch as Bowser Jr.'s UFO carries her off.

In the Mushroom Kingdom, where they now live, Mario and Luigi are celebrated for having beaten Bowser and rescued both the kingdom and Brooklyn. The Tostarenans summon the brothers to investigate a strange disturbance at the Inverted Pyramid in Tostarena Town. Inside, buried under rubble, they find a Warp Pipe with something lodged in it. Their attempts to seal the pipe fail and the creature breaks loose; the brothers ready their Fire forms, but it shrinks back in terror, and Mario, realizing it means no harm, coaxes it into the open. The culprit turns out to be Yoshi. Yoshi had hatched in the Brooklyn sewers — he was meant to be Bowser's wedding gift to Peach before a Bomber Bill detonated in the Warp Zone and pulled pieces of the Mushroom Kingdom, Yoshi among them, into Brooklyn. He had climbed to the surface and explored the city until he toppled a fossil at a natural history museum, after which guards and police hounded him until he fled back through a pipe. The language barrier keeps the brothers from following any of this. After they introduce Yoshi to the Tostarenans, he wanders off; the brothers assume he has abandoned them, but he reappears riding Luigi's bike and travels home with them.

Elsewhere, Bowser Jr. brings Rosalina and the captured Luma to a half-finished planet, intending to free his father from the Mushroom Kingdom now that he holds a princess. Rosalina manages to release the Luma, who escapes to seek out Princess Peach before Bowser Jr. pins her to a chair with his paintbrush.

Some time later, Mario, Luigi, and Yoshi get ready for Peach's birthday party. The gift Mario has prepared leads Luigi and Yoshi to deduce, correctly, that he harbors feelings for Peach he is too timid to voice. At the party, the brothers introduce Yoshi to their friend Toad, who takes an immediate dislike to him. While the others enjoy the celebration, Mario finds Peach alone on the castle roof. She confides that she dislikes birthdays — and that the day is not truly her birthday at all, but the anniversary of the day the Toads found her, leaving her forever wondering where she came from. Mario gives her the gift, a new parasol, to lift her spirits. Soon afterward, Star Bits rain down and a comet crashes into the kingdom: it is the Luma that fled Bowser Jr. Peach and Toad bring it inside, where it pleads for help rescuing Rosalina. The Toad council forbids Peach from going, but Mario stands up for her right to choose. Peach accepts the Luma's plea, Toad gathers supplies, and the Luma becomes a Launch Star that flings the pair to the Gateway Galaxy.

The next morning, the Toads discover Peach missing and fall into a panic. To settle them, Mario, Luigi, and Yoshi take over her responsibilities — guarding the kingdom and rehabilitating Bowser, who remains tiny from the blue mushroom Peach used to shrink him and now lives in a miniature castle, passing his days painting and wrestling with his temper. Luigi warms to him while Mario holds onto his grudge over Bowser's past. No sooner have they checked on him than Peach's Castle is seized by Bowser Jr.'s UFO. The trio evacuates every Toad before the castle is hauled into space. Bowser Jr. confronts them and demands his father's release; when Mario refuses, the boy uses his magic brush to assume his Wonder form, and a fight erupts, briefly interrupted by Luigi nervously trying to introduce himself. Kamek joins in aboard Megaleg, only to be swallowed into an egg by Yoshi, who seizes the controls when Bowser Jr. tries to kill him. Dodging an attack, Yoshi strikes a button that hurls Megaleg and both villains back into the UFO; Megaleg is destroyed on impact and the UFO powers down, dropping the castle into the flower fields of the Honeyhive Galaxy. Stranded there, the trio finds itself saddled with Bowser, who learns for the first time in years that he has a son out there.

At the Gateway Galaxy, Toad notices a missing-persons sign bearing Rosalina's face, whom Peach faintly recognizes. The monkey Ukiki, feigning helplessness, tricks them and steals their supply pack, fleeing to Wart's Casino in the Minus World. There, the owner, Wart, hints that he knows where Rosalina is. After Peach and Toad fight their way through Birdo, Clawgrip, Mouser, a horde of Ninji, and finally Wart himself, Peach swells to giant size with a Super Mushroom and wrings the answer out of him: Rosalina is held at the Space Junk Galaxy. Toad recovers his pack from Ukiki. Furious at his loss, Wart orders Birdo to get Bowser Jr. on the line. The pair surfaces and heads for the Flight Deck — guided by a R.O.B. at the information booth — hoping to hire a pilot, but none will fly them there. Just as their hopes fade, they meet Fox McCloud, a pilot and leader of the Star Fox team from another universe, who crash-landed when his warp drive exploded. Once his story checks out, Peach and Toad hire him, and he readies his Arwing.

On the Honeyhive world, Luigi asks Mario to restore Bowser to full size so the king can find a way to reach Peach. When Mario brings up the attack, Bowser confesses he has been a poor father and wants to mend things with his son; Mario assures him the boy still loves him but refuses to enlarge him. Bowser goads Mario with insults until, enraged, Mario hurls him down and stomps him — returning him to full size. To Mario's surprise, Bowser holds to his reform. The group seeks out the Honey Queen to request passage to the Gateway Galaxy, but she recognizes them as the intruders who wrecked her garden and nearly imprisons them. Bowser surrenders his own freedom to cover their crime, offering his strength as a laborer. The queen agrees; Mario forgives and thanks him before he, Luigi, and Yoshi are carried to the Gateway Galaxy on a Mandibug. As Bowser serves his sentence, the UFO abducts him. Reunited with his son, he is impressed by the boy's work: Bowser Jr. is constructing Planet Bowser, a haven where the Koopas can have everything they were denied in the Mushroom Kingdom, powered by Rosalina's drained energy feeding a planet-destroying cannon called the Boomsday Weapon — inspired by a bedtime story Bowser once told him. Kamek then relays Wart's warning about Peach and Toad.

The three brothers regroup with Peach and Toad and report Rosalina's location, and the pair introduces them to Fox, who takes a quick liking to Mario. Fox offers to fly everyone to the Space Junk Galaxy, but en route they are ambushed by Bowser Jr., who turns Mario and Luigi into infants with a Super Scope and sends the Arwing crashing into Fossil Falls. Bowser feels guilty for betraying the brothers, but his son persuades him that they have dulled his edge, and Bowser resolves to be King of the Koopas once more. In Fossil Falls, while Fox struggles to fix the Arwing, the infant brothers toddle off and stumble onto a sleeping Tyrannosaurus. Yoshi climbs a branch to recover the Super Scope while Toad tries to keep the babies from rousing the beast; Yoshi wakes it anyway, and the group flees on his back. Toad is turned into a baby during the chase. Yoshi shakes the dinosaur when it crashes through a fragile fossil bridge into water, and as it surfaces the babies blast it into an infant with the Super Scope; Yoshi then uses the device to restore Mario, Luigi, and Toad to adulthood. As Fox works on, Mario comforts a discouraged Peach. Luigi idly tests a radio, which reaches the Lumas re-enacting their story in the library; they bring the Comet Observatory to the group, pilot it toward the Space Junk Galaxy, and promise to arrive before nap time.

While aboard, Peach explores the library and finds a storybook about herself, uncovering her origins. Long ago, she and Rosalina were sisters born from stardust on a lifeless planet, Rosalina the elder and Peach the younger. As children they roamed space together and, by pressing their hands together, used their shared power to bring nature and life to their world. One day an unknown force attacked their home in violet light. To save Peach, Rosalina sent her through a Warp Pipe in a cave while holding off the enemy. Peach landed in the Mushroom Kingdom, where the Toads adopted her and later made her their princess. Too young at the time to remember, Peach had carried no memory of Rosalina or her past.

At the Space Junk Galaxy, Fox steers the observatory through the airship armada while Mario, Luigi, Peach, and Yoshi drop onto Planet Bowser to slip in and free Rosalina. The team splits up: Luigi and Yoshi find Koopas loading power-ups, while Mario and Peach are captured and thrown into a trench rigged with Fire Bars, Grinders, Thwomps, and Spike Traps from Bowser Jr.'s security system. The two escape by using Peach's parasol to launch themselves over the wall of Thwomps, boosted by a Bob-omb blast. Refusing to let them break his promise to his son, Bowser confronts Mario and Peach on a bridge above a lava moat. Mario takes one of Bowser's axes and cuts the bridge, plunging Bowser into the lava as Bowser Jr. watches in anguish and Mario and Peach feel a pang of regret. But Bowser survives, rising from the lava as Dry Bowser. Mario sends Peach ahead to reach Rosalina while he, Luigi, and Yoshi take on the Bowsers, transforming with power-ups into Drill Mario, Cloud Luigi, and Blimp Yoshi. As the brothers are stripped of their forms, Blimp Yoshi knocks the paintbrush from Bowser Jr.'s grip; Luigi grabs it and paints Mr. Game & Watch, who fells Dry Bowser with a hammer blow to the head. Enraged, Bowser Jr. retrieves his brush by flinging Dry Bowser's shell at Luigi, taking out Game & Watch in the process, and paints a copy of the Ruined Dragon — which promptly swallows him whole. Mario uses a Red Star to become Flying Mario and defeats the dragon to save Bowser Jr. Meanwhile, Peach finds Rosalina dying at the weapon's core, her energy nearly spent. Remembering their power, Peach urges her to press their hands together from her side; the shared power revives Rosalina at the brink of death and frees her, and the reunited sisters transform Planet Bowser into a clean, living world.

The Lumas reunite with Rosalina and ask to visit Peach's Castle, to the brothers' quiet dread. Returning to the Mushroom Kingdom, they find the castle still entirely gone. Pressed for an explanation, Luigi nervously tells the Lumas the castle has "died," while Mario declares they will rebuild it together — Rosalina and the Lumas pitching in. When it is finished, Mario uses a Cape Feather to become Cape Mario and plant the flag at the peak. Peach kisses him on the cheek to the delight of the Toads before he soars up to the top in elation.

In a mid-credits scene, Fox deposits the Bowsers at a distant prison, his Arwing fully repaired thanks to Rosalina, and heads home. In their cell, the Bowsers try to tunnel out, watched over by their guard Lumalee, who torments them with her relentless nihilism. In a post-credits scene, Ukiki robs a Whittle at the Gateway Galaxy, only to be punched away by a gloved fist; Princess Daisy returns the grateful Whittle's belongings.