After making the perfect plans to spend spring break with Bailey (Debby Ryan), Cody (Cole Sprouse) ditches the plans and goes off to work as an intern at a prestigious research firm, leaving Bailey infuriated and refusing to speak to him. Meanwhile, Zack keeps asking Cody for Cody's car, which his mom and dad will get Cody when he goes to college. But after Zack (Dylan Sprouse) runs a billion-dollar submarine into Vancouver's seawall, and Cody does the same trying to stop him, the equipment is lost and Cody is kicked out from the program and fired from his internship, vowing to never again speak to Zack.
Dr. Olsen (Matthew Glave) approaches the boys and tells them that they would be ideal for the Gemini Project, a massive project studying the effects of twins. Though it takes them much thought, they eventually decide to participate, and land themselves among dozens of other twins. They start out with physical and mental tests, which end in disasters. After eating a special fruit given to them by the researcher, they start to feel each other's physical feelings. Later, they become emotionally similar. After overhearing the doctor speak to the other twins about a "merge," they decide to break into the research office and find out that the Gemini Project is meant to control minds. They decide to run away, but they are seen by the doctor.
Meanwhile, London ate some fruit given to the dolphins, which consequently allowed her to comprehend whatever an animal says because London becomes enabled to hear the dolphins communicating in the English language. Because of London's communication with the dolphin in Dr. Spaulding's tank, she discovers that Zack and Cody are at the Gemini Project 50 miles away. Alarmed, she tells the gang and Bailey calls Mr. Moseby to the site of the Gemini Project, while they all go to save Zack and Cody. During this time, Zack and Cody attempt to run away from the mind controlled twins when they realize they can now communicate telepathically and try to battle each other, but end up just hurting themselves. They are captured, taken to the office, where in a few minutes, Woody, London, Bailey, and Dr. Spaulding came. The doctor makes a shield, so that while Zack and Cody go through the third task, London and the others cannot try to help them. The doctor eventually reveals that he is Dr. Spaulding's evil twin, since earlier in the movie Dr. Olsen says he has a twin.
London and the others find out that he has been spying on Cody, and has been pretending to be the janitor at Dr. Spaulding 's project. Finally, after telling each other off, Zack and Cody become one, but are stopped, because of what they do best - fight. Their fighting ends up breaking Dr. Olsen's project, and he then says he will kill all of them, but is stopped by the police. With Cody's quick thinking, he tells Zack to get the special fruit. After Zack gives the fruit to Dr. Spauldng, Dr. Olsen and his twin feel like brothers again by telepathically finding out that they wanted to be each other. Zack and Cody now understand that they make a pretty good team.
In the epilogue, London brags about saving the day and Zack is driving his car, which Cody let him have, when he reads a sign that says "Do not park, shipping equipment exempted" but interprets it as "Do not park, Zack Martin excepted". Zack parks there and sees his friends and brother on the ship when there's a bang as a gigantic box containing London's summer clothes lands on the car. While Zack lets out a cry, the movie ends with Mr. Moseby saying "Spring break is over, now if I could just make it to summer vacation."
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