Usagi first appeared in the Season 3 episode "Usagi Yojimbo", when Donatello's interdimensional device malfunctioned due to interference from Shredder's device. Upon entering the Turtle's dimension, Usagi assumed that the Turtles were Turtle Guards sent to attack him. Luckily, Michelangelo was able to subdue him by slapping him in the face with a pizza. They decide that Usagi should be returned to his home and that he should stay with the Turtles until they can transport him back.
Usagi helped the Turtles find the source of the disruption. After fighting some Foot Soldiers, Usagi wandered into a rabbit-themed restaurant where he was mistaken for a waiter. Shortly after, he decided to "liberate" his "enslaved brothers," unaware that they were actually humans in rabbit costumes.
After his rebellion, Michelangelo found him and took him to the rest of the group. When Baby-San was hatched, Usagi was part of the group that found Obento. Usagi was the one to place the Blue Flame of Osaka in the dragon's mouth, causing him to revert to his egg form. Afterwards, Obento agreed to care for Usagi until he could return home.
In the episode "Usagi Come Home", the Shredder had plans for the samurai rabbit, by kidnapping Obento and fighting an unfair duel with Usagi that left the rabbit honor-bound to attack the Turtles. Usagi attempted to kill the Turtles in the ensuing fights, until Shredder's dishonorable tactics (a giant magnet that drew away Usagi's sword) were revealed.
Usagi helped the Turtles infiltrate Shredder's new hideout, just as Shredder chose to betray the rabbit warrior by trying to force him to continue being in his employ. The Turtles interrupted by flooding the building with fireworks, and Usagi freed Obento before he and his friends fled the place.