He is voiced by Darren Dunstan who also voices Maximillion J. Pegasus from Yu-Gi-Oh!.
Splinter is depicted as having gray fur instead of the brown fur of his other incarnations.
He does not wield a traditional Japanese weapon, Splinter carries a cane which he can utilize as a weapon (like a Jo staff) with fierce proficiency.
Splinter is a wise and loving father, treating the turtles as his own sons but not hesitating to scold them when necessary. He does his best to look out for them and would gladly give his life to protect them. In an alternate future where the Shredder takes over the world, that is exactly what he did. This overprotective streak made him reluctant to allow them to enter the Battle Nexus competition; in fact he never even told his sons about it and they only found out because they followed him. However Splinter also shows great faith in his sons, believing them to be fully capable ninjas and even showing enough confidence in their abilities to allow them to face the deadly Shredder on multiple occasions, although during the majority of their encounters, he is there to support and protect them in any way that he can.
Splinter has a vengeful streak as shown by his determination to avenge Hamato Yoshi's murder. He speaks very highly of Yoshi, even thinking of him as a father figure despite the species differences and the fact that Splinter wasn't fully sentient until after Yoshi's death. He is one of the very few characters who challenges the Ninja Tribunal, whom he partially blames for Yoshi's death. Indeed Splinter shows blatant contempt for the Tribunal, believing them to be heartless and no better than the demon Shredder. However, after the demon Shredder is destroyed, the Tribunal explained that if Yoshi never died, he would never have existed and neither would the turtles.
Even before his mutation, Splinter showed a remarkable level of intelligence for a rat, being able to mimic Master Yoshi's movements and understand the principles of ninjitsu enough to retain and teach them to his sons. Despite Splinter's intelligence however, he has great difficulty using mechanical equipment, being unsure of how to answer a Shell Cell or control the Battleshell remotely.
Splinter is a very honourable person and is therefore disgusted by dishonour and cowardice. He is capable of fighting on par with the Shredder and also shows a great deal of cunning and resourcefulness in combat, such as when he tricked the Shredder into bringing a water tower down on himself. Despite his age, Splinter also shows amazing speed, strength and skill in combat. His simple walking stick is his signature weapon as he believes that in the hands of a true ninjitsu master, any weapon could be deadly.
In addition to his exceptional skill in physical combat, Splinter is by no means lacking in other aspects of ninjitsu. He was able to penetrate the illusion created by the Foot Mystics and he and his sons defeated them by using meditation. Splinter's keen sense of observation is assisted by his strong sense of smell. Even when his opponents are invisible, he can still pick up their scent and get at least a vague idea of where they are.
He once was a pet for a renowned martial artist named Hamato Yoshi in Japan. It was from watching Yoshi that Splinter first learned the art of ninjitsu which he then passes on to the Turtles.
Splinter escapes from his cage during the murder, and attacks Saki, clawing at his face. Saki in turn slices his ear off. It is suggested that this mutilation is the reason Saki took the Shredder disguise to hide the scars. In contrast, in the 2003 TMNT cartoon it was Hun's face who Splinter clawed.
Without a home, Splinter is forced to run away and live in New York City's sewers. Due to a traffic accident, four baby turtles and a canister of radioactive ooze are sent down into the sewer. The canister mutates both Splinter and the turtles. Splinter names the four turtles Leonardo, Donatello, Raphael, and Michelangelo (after a book on Renaissance artists that he found in a storm drain) and trains them in the arts of ninjutsu, so that they can take revenge for his dead master, while raising them as his own sons.
He possesses a significant reputation as a ninja master; in a multi-part episode, he is revealed to be a champion of the Battle Nexus, a multi-dimension-spanning contest where the greatest combatants of various dimensions come together to fight each other for the title of Battle Nexus Champion. When the Turtles learn of the Battle Nexus, they participate as well, resulting in Splinter forfeiting when called upon to fight Michelangelo, as he wishes to allow his sons the opportunity to fight where he succeeded in the past.
In the 2003 episode, "Fast Forward", Splinter is sent to the future with the Turtles, but the villain, Viral, blasts Splinter with a decompiler ray that scatters his bits all over the internet. While Splinter wasn't seen much in the "Back to the Sewers" season, the focus of those episodes is the Turtles' efforts to find Splinter's data bits by traveling all over cyberspace. Splinter is restored in the final episode and helps defeat the Cyber Shredder.
- Unlike the previous animated series, Splinter and the turtles have more of a "father-son" kind of relationship rather than "mentor/teacher-student" relationship, as while the turtles often call him "Splinter," "Master" or "Sensei," they consider Master Splinter their dad while Splinter considers the turtles his sons.