The main film's setting is in the water-themed Johto city of Altomare (which means "High Sea"), based on cities such as Venice, Italy, Amsterdam, Netherlands and Bizerte, Tunisia . The city is watched over by two legendary Pokémon, Latias and Latios. The story behind the two involves an evil Pokémon Trainer using a Kabutops and an Aerodactyl to terrorize the citizens, until the original Latios came to the city, using his powers to drown the evil Pokémon and turning the streets into canals. However, Latios died and his children were left orphans. His soul is within a special jewel, the Soul Dew.
The Soul Dew is sought by Annie and Oakley, two members of Team Rocket. The Soul Dew acts as a key for activating the Defense Mechanism of Altomare, a special device built in case of disaster. Meanwhile, Ash Ketchum, Misty, and Brock are visiting Altomare on their travels, along with Hubie, Marina, Rocko, Splatter, Petra, Beanie, Timmy, Pikatwo, Bonkers, Fawn, Buck and Stanley. Ash and Misty participate in the Tour de Altomare, a water chariot race. Misty wins, after Ash takes a wrong turn due to an invisible Latias and Latios. Ross, the former champion of the race gives the friends a tour of the city in his gondola and tells them of the city's guardians. Annie and Oakley follow Latias, disguised as a human girl named Bianca. Latias is saved by Ash, Hubie, Marina, Pikachu and Pikatwo. Team Rocket are also in the city and decide to follow Annie and Oakley.
The team later visit Lorenzo, the curator of a local museum, who the city's history and also introduces them to the Defence Mechanism of Altomare (DMA). Ash, Hubie, Marina, Pikachu and Pikatwo pursue Bianca, and then Latias in disguise, across the city, eventually discovering a hidden garden where Latias and Latios live. When Latios and Bianca try to attack them, Lorenzo arrives to clear up all the tension. Latias and Latios play with Ash, Hubie and the two Pikachu, and later Lorenzo reveals to them the Soul Dew.
Later on, Bianca takes the group to the Pokemon Center. But Marina is angry at Bianca for nearly attacking her husband and while the others aren't looking, she threatens her, saying that if she tries to hurt her family, she'll pay. Bianca feels heartbroken and thinks of a way to make it up to her. Ash eventually figures out what Marina has done and the two argue a bit before Ash tells Marina to reconsider.
However, Annie and Oakley's spy robot had been watching the whole playtime and the reveal of the Soul Dew and later the duo steal it and capture Latios using both to control the DMA. Latias flees to Ash, Hubie and his friends for help, and later Ash, Latias, Hubie, Marina, Rocko, Pikachu and Pikatwo race across the gridlocked city to the museum. Using the DMA, Oakley locks down the city and revives the undead Pokémon to capture Latias. After a long chase through the city, the team cause the undead Aerodactyl to crash into a building, and are saved from the Kabutops by their friends' Pokémon. The heroes arrive at the museum to find the DMA going berserk and Latios seriously injured. After freeing Latios, the Soul Dew fades and turns black, causing the DMA to shut down. When Annie tries to get the Soul Dew, it disappears and the city's water becomes a giant tsunami. Latios and Latias use their psychic powers to stop the tsunami, but Latios sacrifices himself in the process. The water returns to the city, washing away Team Rocket in the process. The undead Pokémon return to their fossil states, and Annie and Oakley get trapped inside the DMA.
Latios becomes a new Soul Dew and is placed as the new guardian of Altomare. In light of the events, Marina apologizes to Bianca for her behavior towards her, stating she wasn't herself.
The heroes head off to leave Altomare, but Bianca or Latias in the guise of Bianca (Nobody knows which) stops them before they leave and hands Ash a drawing of him and Pikachu. She gives Ash a kiss on the cheek before she walks up to Marina and give her a drawing too and leaves. It's a picture of her and Hubie and a message at the bottom saying, Take good care of each other. Bianca.
As the group leave on a boat, they see two Latios and a Latias in the air. Annie and Oakley are sent to jail, where they examine the possessions of Lawrence III.