- Cast overview, first billed only:
- Tobey Maguire .... Peter Parker/Spider-Man
- Kirsten Dunst .... Mary Jane Watson
- James Franco .... Harry Osborn
- Thomas Haden Church .... Flint Marko/Sandman
- Topher Grace .... Eddie Brock
- Bryce Dallas Howard .... Gwen Stacy
- Theresa Russell .... Mrs. Marko
- Bill Nunn .... Joseph Robertson
- Tanya Sinovec .... Jazz Club Waitress
- Dawn Marie Anderson .... Nurse
- Robert N. Anderson .... Lab Technician
- Terrell Anthony .... Lab Technician
- Elizabeth Banks .... Betty Brant
- Jodi Benker .... Woman in Nightclub
- Wilson Better .... Chess Bully
