Princess Rosalina, Brie Larson, who lives in the Comet Observatory and serves as the adoptive mother to the Lumas, is kidnapped by Bowser Jr., Benny Safdie, who plans to drain her power to fuel a universe-destroying cannon built in honor of his father, Bowser Jack Black. A Luma escapes and travels to the Mushroom Kingdom, where Peach Anya Taylor-Joy and Toad Keegan-Michael Key answer the alarm and enlist help. Mario Chris Pratt and Luigi Charlie Day head to the desert town of Tosterena, where they befriend a friendly dinosaur named Yoshi Donald Glover. Together, the trio—along with Yoshi—protect the Mushroom Kingdom in Peach’s absence and work to rehabilitate Bowser, hoping to bring his power back under some form of control.
Bowser Jr. lifts Peach’s castle into space in a bid to free his father, but the plan backfires when the castle is accidentally dropped into the Honeyhive Galaxy. Bowser finds himself unshrunk and offers to surrender in exchange for the brothers and Yoshi, only to be abducted by his own son, whom he hadn’t seen in years. In the Gateway Galaxy, Peach and Toad search for Rosalina after re-taking Toad’s backpack from Ukiki, who is working for Wart, a frog-like casino owner; Wart’s influence helps them zero in on Bowser Jr.’s location.
Mario, Luigi, and Yoshi reunite with Peach and Toad, who have hired pilot Fox McCloud Glen Powell to transport the group. Fox’s spaceship crash-lands in Fossil Falls after Bowser Jr. launches another assault, and Bowser Jr. uses a Super Scope to briefly turn Mario and Luigi into babies. Bowser expresses some regret about bonding with the brothers, but Bowser Jr. convinces him to resume his kingly duties over the Koopas. Yoshi locates the Super Scope, rescues the brothers from a Tyrannosaurus rex, and restores their ages, while Luigi signals the Lumas to ferry everyone back to the Comet Observatory.
Back in orbit, Peach discovers she shares powers with Rosalina—revealing that Rosalina is her long-lost older sister, who sent an infant Peach to the Mushroom Kingdom to protect her from an unidentified attacker. The crew uses the Comet Observatory to send the others to Bowser Jr.’s hideout and prepare for a final confrontation. Mario and Peach face Bowser in a climactic duel; Bowser is driven into a lava moat and appears defeated, but he survives in a skeletal form, and Luigi and Yoshi join Mario in a final push with power-ups. Peach’s steadfast resolve helps rescue and reunite Rosalina with the Lumas, and Peach’s castle is rebuilt as Fox dispatches the Bowsers to prison and heads home to his own galaxy. In a separate moment of courage, Princess Daisy prevents another robbery by Ukiki in the Gateway Galaxy, keeping the wider cosmos safe for now.
