The movie begins in feudal Japan, 1603, with a masked man fighting four samurai on horseback. As the samurai ride off with the man, a mysterious woman emerges from the underbrush and tries to help him. However, the samurai eventually capture and take the man with them.
Fast forward to present day Manhattan, New York (1993), the scene switches to Pooh And The Others & the Teenage Mutant Ninja Turtles who still live in the abandoned subway station. During their practice, the Turtles are met by April O'Neil, who has been shopping in preparation for her upcoming vacation and brings her friends gifts to cheer them up. Among those items is an ancient Japanese scepter, which was supposed to be for Master Splinter.
In feudal Japan, the masked man resulted to be Kenshin, son of the Daimyo Lord Norinaga. Angry against his father's unfair war against a group of rebel villagers, Kenshin leaves his father's presence and drives priests out of a temple. There, he finds the same scepter and reads the inscription on its handle: "Open Wide the Gates of Time". Before Kenshin leaves, a British weapons-trader called Walker is introduced, along with his thug Niles.
As the scepter begins to emit winds and strange lightning, April and Kenshin are switched in their respective times exchanging clothes. Upon arrival, April is captured by the Daimyo's soldiers. Lord Norinaga blames her for his son's vanishing and sends her to dungeons.
The Turtles become friends with Kenshin and decide to travel back in time to retrieve April. They invite Casey Jones to watch over Splinter, then embark, warned by Donatello's calculation that they have only 60 hours (2 and a half days) wherein to complete the rescue before the scepter ceases to enable time travel due to the time-space continuum being out of sync. Having been sent to Japan in exchange for Daimyo Norinaga's Honor Guards, the Turtles arrive mounted on horses in the midst of a battle. Michelangelo is separated from his brothers and captured by outlaws. The scepter goes with him and is ultimately seized by an unseen character. When the other Turtles search for April at Norinaga's fortress, they are quickly mistaken for Honor Guards, then follow Walker's men to the prison and take out April through a garbage-disposal chute. They also help an Englishman called Whit who is said to have rebelled against Walker, and who bears a strong physical resemblance to Casey Jones (both parts are played by Elias Koteas).
An attack on the Turtles, who were again mistaken for the Honor Guards, leads them to Mitsu, leader of the rebellion against Lord Norinaga. She is a young, beautiful, courageous, and determined woman who intends first to overthrow the Daimyo and later to marry Kenshin so as to prevent another war. It is she who has imprisoned Michelangelo. Mitsu's village is under attack by Walker and his men, who are searching for the scepter; therefore the Turtles rush to intervene. Having seen his face and heard him speak, they plant the rumor among their partners that a demon is in the vicinity. When confronted by Michelangelo, the other Turtles, and Mitsu's people, Walker flees. Michelangelo saves a boy named Yoshi from a fire, whereupon Leonardo revives the nearly dead child by means of CPR, for which the grateful villagers allow them to stay. Anguished by the loss of the scepter, the Turtles commission a local blacksmith to make a new one.
Back in Norinaga Castle, the Daimyo reveals a legend to Walker stating that four kappa resembling the Turtles, had vanquished his ancestors, and suggests that they have returned to disgrace him. Walker offers to use his cannons to destroy "these monsters" if Norinaga accepts to pay for the weapons.
Michelangelo is apparently attracted emotionally to Mitsu, and reassures her that Kenshin will return when the Turtles have departed. Raphael, meanwhile, fulfills his potential patience and gentleness through Yoshi. He becomes very fond of this child and eventually considers him a younger brother.
The new scepter is completed, only to be broken by Michelangelo and Raphael during an argument. Mitsu then informs them that Lord Norinaga has guns and will attack the next day. Yoshi, fearing Raphael's death, gives him the original scepter, which he had hidden under his grandfather's command, because the latter wanted the Turtles and not Mitsu to fight the Daimyo. Mitsu is then kidnapped by Whit, who resulted to be Walker's spy and steals the scepter to Walker.
The Turtles set out to rescue Mitsu. In the process, they learn of the legend that has caused the Daimyo to fear them. They free many of those imprisoned, leading to a massive battle in the courtyard of Norinaga's palace between the rebels and the Daimyo's army. Leonardo defeats Lord Norinaga in a sword duel, comically finishing him by cutting his hair (a disgrace to his status as a samurai), and then trapping him inside of a bell.
Walker, at the end of the battle captures April and threatens to kill her. His soldiers then take control of the battlefield, armed with guns. He orders the Turtles to be shot, whereupon Leonardo bluffs by drawing upon the belief (held insistently by Walker's superstitious men) that he and his brothers are demons, saying that any bullets shot at them will ricochet and kill their senders. Baited by Leonardo to take the shot himself, Walker shoots at them with a cannon, but misses when Leonardo instinctively pulls his head into his shell. April and Whit, also targeted by the cannon (Whit having spoken against Walker's order), duck their own heads to dodge the shot. The cannonball collides with the bell, freeing the shocked but largely unscathed Daimyo.
Walker's soldiers are routed and he himself runs away. He obtains the scepter and a cage containing pet birds from his room, then climbs over the roof of the palace to a high point overlooking the ocean. He is there cornered by the Turtles. To distract them while escaping himself, Walker throws the scepter overhead and begins climbing down a scaffold. The Turtles form a chain with their bodies, holding onto a wall or fence at one end and catching the scepter at the other. Walker, realizing that he has forgotten his birds again, returns to retrieve them, whereupon Whit, realizing that his name is "lower than scum" in the eyes of the desirable April, uses a catapult to kill Walker by knocking him to his death in the ocean.
The Turtles then debate whether or not to go home, wishing to remain on grounds that they are appreciated and respected in feudal Japan, unlike 20th-century New York City where they must hide underground to avoid being targets of human xenophobia. Mitsu urges them to go, so that she may have Kenshin return to her, and makes a parting well-wish to Michelangelo, of whose soft spot for her she is aware and by which she is flattered. The scepter then activates, making their decision urgent. In New York City of the late twentieth century, Kenshin has activated it in his impatience to go home. Splinter counsels him not to abandon the Honor Guards, on the grounds that such an act would be cowardly. Casey gathers the Honor Guards, who have become infatuated with the trappings of their host society, and organizes their return to feudal Japan.
The Turtles ultimately decide to go home, based on the argument that their presence interferes negatively with the lives of the people of feudal Japan. Raphael bids Yoshi an affectionate farewell. As a result of his procrastination, Michelangelo fails to join April and his brothers. When they have returned to their native time, the Honor Guard who had replaced Michelangelo runs away, carrying the scepter. As he exits the Turtles' den, the scepter activates, exchanging him for Michelangelo. Simultaneously, its powers of time travel (symbolized by the rotating device at its head) are destroyed. In Japan, Norinaga is taken prisoner and brought before Mitsu and Kenshin. He is surprised to see them both together, and more surprised to see his Honor Guard walk through the corridor, half naked and talking incoherently about anachronisms. When he has gone, having spoken no word but clearly shown his submission, Kenshin seizes Mitsu in imitation of a poster he had seen in the Turtles' home and kisses her on the lips. Norinaga, presumably is reconciled with his son.
Michelangelo, perhaps thinking on the preceding scene, is depressed until Splinter puts the afore-mentioned lampshade on his (Splinter's) head as a joke about Elvis in Hawaii, whereupon both laugh. The film ends with another dance sequence by the Turtles..