The Turtle Sub was a submarine that Donnie made so that he and his brothers could go to the underwater Kraang base. It first appeared in the episode Karai's Vendetta. The sub resembles a sea turtle and is equipped with engines that are charged by kinetic energy. This makes it necessary for the other three turtles to pedal on exercise bikes to charge them while Donnie navigates. The sub is also equipped with weapon systems including flares and depth charges. The "beak" of the sub is very strong and is capable of clamping onto large objects and moving them out of the sub's path. For the nagivagtion, the sub is equipped with a toilet seat periscope.
- In the episode Karai's Vendetta, the Water Creature thought the Sub was a female and tried to mate with it, disturbing the Turtles.
- The Turtle Sub bears a resemblance to a sea turtle.
- Playmates released a Turtle Sub vehicle with a Diver Donnie figure.
- It makes another appearance in Plan 10, with Casey and April taking over Raph and Mikey's place.