The film begins with the vigilante quartet of anthropomorphic turtles—Leonardo, Raphael, Michelangelo, and Donatello—continuing their efforts from the shadows to protect New York City. The brothers strive for a normal life above ground but must remain hidden due to being outcasts despite their heroics. Their friend, April O'Neil, uncovers a plan by the villainous scientist, Baxter Stockman, to break the nefarious Shredder out of prison while being transported between facilities. The breakout attempt escalates when Shredder is inadvertently teleported to another dimension by a teleportation device and comes into contact with the manipulative alien warlord Krang. Krang provides Shredder with a mutagen that has the power to transform humans into animals, which he plans to use to build an army of mutated henchmen.

Shredder returns to Earth and recruits two criminals, Bebop and Rocksteady, transforming them into a warthog and a rhinoceros through the mutagen's power. Meanwhile, the turtles face their own internal conflict about using the mutagen to become human and integrate into society. At the same time, they also must stop Shredder's plans. They're assisted by their rat sensei Splinter, the vigilante Casey Jones, and their old ally Vern Fenwick. As the threat of Krang's impending invasion looms with his Technodrome, a massive battle station capable of destroying entire cities, the turtles must confront their desires and fears to save the city from complete destruction.