Usagi is a white, anthropomorphic rabbit, with a curved scar above his left eye. His ears are tied back with blue material in the shape of the traditional samurai topknot. He wears a blue kimono, and has his embroidered crest of his old clan on his chest. The same sign is also carved from wood and attached to his sash. His hakama are navy blue. He also wears white socks with waraji straw sandals. He carries two swords, a traditional katana and wakizashi.
Miyamoto Usagi was Lord Mifune's personal bodyguard in the battle against Lord Hikiji. Usagi believes his forces might have been victorious, had not a trusted general turned against Mifune. With Usagi having failed to protect his Lord's flank, the general killed him. In Usagi's grief, he became a ronin, roaming the land of feudal Japan, seeking to redeem himself.
In his debut episode in the series, Usagi is first seen just after witnessing the wolf-demon Jei having murdered a family. Usagi berates Jei on how he could do something like that, calling him insane and a demon. Jei claims that his actions were justified, saying that they were infected with evil and the gods revealed their nature to him. Usagi proclaims that he has tracked him down to bring him to justice. As Usagi and Jei do battle, Jei continues to claim that the gods gave him his skill to cleanse this realm of evil, to which Usagi replies that he is the evil one. Just before Jei strikes him down, Usagi asks him who he is, to which he replies "I am Jei, the Blade of the Gods" and then disappears as he is struck by lightning. He comes across a ransacked village and meets an extraordinary child by the name of Kintaro. He is tasked with protecting the boy from Jei until he finally realizes his own inherent superhuman abilities at the sacred Temple Palace. Usagi accompanies the boy Kintaro, blessed with divine powers, on his way to a safe place until Jei brings the Turtles into their world to kill Usagi for him. While setting camp, Usagi is ambushed by the Ninja Turtles who are controlled by Jei's evil magic. But after the Turtles are defeated by Usagi, they make friends with the Ronin. He welcomes the new comers and explains his history as a samurai of great strength and skill. The Turtles accompany him on his quest in the hope that they can find a way back home. Usagi and the Turtles try and pursue Jei's Sumo Kuma but are discovered and fall off a cliff.
In a haunted forest of yokai, Usagi helps the Turtles fend off shapeshifting spirits that capture Michelangelo and Raphael. He appeases them by offering a carrot as a peace offering. Upon entering a cave full of giant spider webs, he is compelled by the voice of his good friend Akemi who is, in fact, a spider.
As Usagi, Kintaro, and the Turtles continue to make their way to the Temple of the Sky Buddha, they soon reach several obstacles along their way, while Kintaro continues to boast and complain that he's better than any of them. They soon are attacked by several cat ninjas that can blend in with the snow and blizzard conditions. The ninja nearly capture Kintaro for Jei, but the Turtles and Usagi fight them off, but it results in Kintaro being injured. While Usagi, Donatello, Raphael, and Mikey start towards the temple, now visible in the distance, Leo remains behind to offer some humility and wisdom to Kintaro about his courage dealing with the cat ninja. Once they reach the temple, Usagi realizes something is wrong as there are no guards to welcome them. When they reach the courtyard, they find Jei waiting for them, along with Sumo Kuma, revealed to be the head monk and leader of the Temple, but was brainwashed by Jei as well. Jei soon manages to brainwash the Turtles again to attack Usagi, but just as the brainwashed Leo is about to deliver the killing blow, Kintaro is able to block the attack, before summoning his true power, which allows him to easily overpower the Turtles and break Jei's spell on them, even overpowering Sumo Kuma as well when he tries to face Kintaro in a sumo match. However, Jei is able to capture Kintaro, but as he begins the ritual to transfer his immortal soul into Kintaro to become unstoppable, Leo runs him through with his katana, causing Jei to realize the Turtles are no longer under his control and use Kintaro against them. This enrages Usagi to the point that he manages to finally defeat Jei, pulling out Leo's katana that manages to imprison Jei's soul within it, leaving Jei's body to plummet to his death, ending his threat for good. As Sumo Kuma comes to and apologizes for his actions under Jei's brainwashing, he promises to train Kintaro well, reminding Kintaro of his power, which he uses to create a portal to send the Turtles safely back home to NYC. His mission complete now that Kintaro was safe with Sumo Kuma and the Temple, Usagi departs to find others in need of his service, remaining loyal to the Bushido code of the samurai as a Ronin.
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- Originally, John Boyega was stated to be the voice of Miyamoto Usagi, but instead the Japanese actor Yuki Matsuzaki was confirmed as the voice just before the release of the episode Yojimbo.
- His appearance was worked on by Ciro Nieli and Stan Sakai himself.