Tobey Maguire (Spider-Man / Peter Parker), Kirsten Dunst (Mary Jane Watson), James Franco (Harry Osborn), Alfred Molina (Doc Ock / Dr. Otto Octavius), Rosemary Harris (May Parker), Donna Murphy (Rosalie Octavius), Cliff Robertson (Onkel Ben Parker), Willem Dafoe (Norman Osborn), J.K. Simmons (J. Jonah Jameson), Daniel Gillies (John Jameson), Ted Raimi (Hoffman), Elizabeth Banks (Miss Brant), Dylan Baker (Dr. Curt Connors), Mageina Tovah (Ursula), John Landis (Doktor), Vanessa Ferlito (Louise), Stan Lee (Mann, der Trümmern ausweicht), Aasif Mandvi (Monsieur Aziz), Bill Nunn (Joseph 'Robbie' Robertson), Scott Spiegel (Mann auf dem Balkon)