Splinter was once a ninja named Hamato Yoshi, before he was expelled from the Foot Clan. He was later mutated into a giant rat man. Splinter is the ninja master and father figure to the Ninja Turtles.
Splinter accidentally swallows a vampiric virus called the Nanovampire, which was planted by Krang. The Turtles are forced to shrink down and enter his body and defeat the Nanovampire to save him.
As the Turtles battle Shredder, Bebop, and Rocksteady, Splinter and the fairy Kris Mu infiltrate the Technodrome. While there, they encounter Krang, and the old rat fights the alien brain.
After New York City is flooded, Splinter rejoins the Turtles and deflects one of Dark Demon Shredder's dark energy balls.
The Turtles, Splinter, and April O'Neil head to Japan to help an old ninja friend of Yoshi's, Hattori Kinzō, exorcise a ghost.