Working as a child actor since his early teens, he appeared in a variety of commercials and TV and movie roles. He auditioned for a part in the series Parenthood, where he met best friend actor Leonardo DiCaprio (DiCaprio got the part although Maguire would appear as a guest star in later episodes). Maguire gained significant attention with his role in the film The Ice Storm, directed by Ang Lee. This soon was parlayed in a variety of lead roles where he played a thoughtful boy coming of age in a variety of films such as Pleasantville, The Cider House Rules and Wonder Boys.

In 2002, he shot to stardom in the title role in Spider-Man, where he was praised for his performance of the troubled superhero. He further solidified his stardom in 2003 with a leading role as the jockey John M. "Red" Pollard in an acclaimed film Seabiscuit, about the famous American racehorse Seabiscuit. Spider-Man 2, the sequel to the original blockbuster, was released in 2004. He will reprise the role of Spider-Man in 2007 with the third Spider-Man movie in the series.

In 2004 Maguire also took up tournament poker. He has finished in the money in several events and has been tutored by poker pro Daniel Negreanu. He is also a vegetarian and practices yoga.