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Luigi was at the Flower Castle with Princess Daisy. She asks him what college he went to and Luigi says he didn't go to college. Toad comes and tells Luigi that Mario and Peach have been kidnapped. Luigi decides to go save them but bumps into Mushroom University on his way. When he wakes up, a police Toad tells him that the kidnapper is in the building, so he decides to go to college.
In this game, Worlds are replaced by colleges, though each one is in a different world.
- 1-1: Sign-Up Sign Up: The mission in this level is to put signs up so Luigi can sign up for college.
- 1-2: This Means Wario: The mission is to beat the other football team which Wario is on.
- 1-3: Substitute Teacher: Luigi must find out the real name of his substitute teacher.
- 1-T: Homework: A giant Reznor takes over the school and Luigi must defeat it.
- 1-C: Junior High: Bowser Jr. takes over the school.
- 2-1: Trans-Furred: Giant hypnotised Fuzzies are out to turn everyone evil!
- 2-2: Flexi-Ball: Luigi has a hard time when Waluigi is the coach for P.E.
- 2-T: Volcano Physics: A giant Venus Fire Trap burns the school.
- 2-3: Sushi Attack: While Dolphin-Riding, the Sushis go into battle with the dolphins!
- 2-Boo: Fright Fest: Ghosts are haunting the school and Luigi must find out who's doing it.
- 2-C: Room Maytee: Luigi gets a new roommate who is Captain Boo, a King Boo from a sea universe.
- 3-1: I Have A Dream: Luigi has a dream about him in the original Donkey Kong trying to save Daisy.