A traveling ballet troupe comes into town and Duck's whole class gets time off to go and see the troupe practice. Duck sees that a heart shard has taken possession of the lead in the upcoming Sleeping Beauty show, but it leaves when the void in her heart is filled when the lead's husband encourages and supports her. Duck chases after the shard and asks it to return to Mytho, but it instead attacks her with a sword. Despite its obvious reluctance, Duck catches it and returns it to Mytho. The shard turns out to be the feeling of fear and uncertainty, and once it is returned Mytho becomes afraid of Princess Tutu. Duck is shocked and dismayed that her attempt to help Mytho has ended with him in pain.
Princess Tutu follows Duck, a duck who was transformed into a young girl and takes ballet at a private school. She becomes enamoured of her mysterious schoolmate Mytho, and transforms into Princess Tutu to restore his shattered heart. Mytho's girlfriend Rue transforms into Princess Kraehe to frustrate Tutu's efforts, and Mytho's protective friend Fakir discourages Mytho's burgeoning emotions. When it becomes apparent that Duck, Rue, Mytho, and Fakir are meant to play out the characters in a story by a long-dead writer named Drosselmeyer, they resist their assigned fates and fight to keep the story from becoming a tragedy.
