At the beginning of the game, Princess Peach holds a surprise party for Mario. When Mario arrives at the party, Bowser comes in his airship and attacks the castle. Mario is hit by a spell cast by Kamek, and he gets knocked unconscious. Princess Peach is then captured by Bowser. Afterwards, Bowser destroys the Shine Orb, scattering the shine sprites inside the sphere. Since the shine sprites were what powered the sun, the Mushroom Kingdom went dark. When Mario wakes up, he meets a toad named Eddie who explains what had happened. Mario sees a shine sprite on the ground, and resolves to collect every one of the missing 64 shine sprites.


Mario collects several shine sprites, and eventually fights Bowser for the second time. After defeating Bowser Bowser explains to Mario that now that he has the Orb of Power he will destroy the Mushroom Kingdom. Bowser explains that the Mushroom Kingdom will be destroyed by a Meteor he summoned. The Meteor will crash into the Mushroom Kingdom, destroying its inhabitants, and any toads who wish to live would have to join Bowser's forces. Later, when Mario travels to Kamek's castle, he learns that the orb of power has the souls of Ancients, tyrants who lived thousands of years ago, who have taken over Bowser's body.


Mario travels to Bowser's castle and gets through it to face off against Bowser. After defeating Bowser, however, it turns out the Bowser Mario faced was just a robot, and Mario needs to defeat the real Bowser. Mario triumphs over Bowser, though, and heads to the top of the castle. At the top of Bowser's Castle, Mario sees the meteor is about to crush the Mushroom Kingdom. After speaking with Kamek, Mario devises a plan in which Kamek makes the Meteor explode in the forcefield protecting Bowser's castle, eliminating Bowser's Castle but leaving the Mushroom Kingdom unharmed. Kamek then offers both Mario and Peach a ride on his broom, but Mario is hit by a stray meteorite, falling down towards the earth. Kamek is forced to leave with Peach, and the tower holding the Orb of Power crumbles. Mario, meanwhile, lands on the side of the tower, and proceeds to navigate through the remains of Bowser's Castle, which is falling apart due to the Meteor of Ultramite Destruction's proximity to the earth. Mario finds a Sling Star, and flies out of Bowser's force field just as the Meteor crashes into the earth. The explosion is safely contained within the force field, and Bowser's Castle is completely obliterated. Mario lands on the Castle Grounds, where Kamek and Peach are waiting. Kamek then departs, but not before reminding Mario that they are still enemies. The Toads then congratulate Mario, and remind Peach about the party for Mario. They all then go inside to have some of Peach's cake, while the credits roll. At the end of the credits, a "The End" is shown on the screen, only for a question mark to appear at the end, reminding the player to collect the rest of the 64 Shine Sprites. Mario is heard thanking the player for playing his game.