After being bitten by a genetically altered spider at Oscorp, nerdy but endearing high school student Peter Parker is endowed with amazing powers to become the superhero known as Spider-Man.
| Spider-Man / Peter Parker | Tobey Maguire |
| Green Goblin / Norman Osborn | Willem Dafoe |
| Mary Jane Watson | Kirsten Dunst |
| Harry Osborn | James Franco |
| Ben Parker | Cliff Robertson |
| May Parker | Rosemary Harris |
| J. Jonah Jameson | J.K. Simmons |
| Flash Thompson | Joe Manganiello |
| Maximilian Fargas | Gerry Becker |
| Joseph 'Robbie' Robertson | Bill Nunn |
| Henry Balkan | Jack Betts |
| General Slocum | Stanley Anderson |
| Dr. Mendel Stromm | Ron Perkins |
| Carjacker | Michael Papajohn |
| Simkins | K.K. Dodds |
| Hoffman | Ted Raimi |
| Ring Announcer | Bruce Campbell |
| Miss Brant | Elizabeth Banks |
| Houseman | John Paxton |
| Philip Watson | Tim DeZarn |
| Madeline Watson | Taylor Gilbert |
| Bone Saw McGraw | Randy Poffo |
| Wrestling Promoter | Larry Joshua |
| Wrestling Arena Guard | Timothy Patrick Quill |
| Bone-ette | Lisa Danielle |
| Bone-ette | Natalie T. Yeo |
| Bone-ette | Erica D. Porter |
| Bone-ette | Kristen Davidson |
| Flash's Crony | Jason Padgett |
| Teacher | Shan Omar Huey |
| Girl on Bus | Sally Levi |
| Doctor | Evan Arnold |
| Nurse | Jill Sayre |
| Project Coordinator | Jim Ward |
| Test Pilot | David Holcomb |
| Check-In Girl | Octavia Spencer |
| Heckler | Brad Grunberg |
| Little Billy | Shane Habberstad |
| Billy's Mom | Deborah Wakeham |
| Times Square Child | Rachael Bruce |
| Times Square Child | Mackenzie Bryce |
| Times Square Child | Julia Barrie |
| Macy Gray | Macy Gray |
| Cop at Fire | Myk Watford |
| Fireman | Bill Calvert |
| Mother at Fire | Sylva Kelegian |
| Young Lady at Fire | Kristen Marie Holly |
| Cabbie | Ajay Mehta |
| Cabbie | Peter Appel |
| Marine Cop | Scott Spiegel |
| Cop at Carjacking | Matt Smith |
| Cop at Carjacking | Sara Ramirez |
| Punk Rock Girl | Lucy Lawless |
| Subway Guitarist | Jayce Bartok |
| Lady Dogwalker | Maribel González |
| Office Lady | Amy Bouril |
| Opinionated Cop | Joseph D'Onofrio |
| Surly Truck Driver | Jim Norton |
| Chaperone in Tram | Corey Mendell Parker |
| Girl in Tram | Ashley Edner |
| Boy in Tram | William Joseph Firth |
| Boy in Tram | Alex Black |
| Tram Group Mother | Laura Gray |
| New Yorker on Bridge | Joe Virzi |
| New Yorker on Bridge | Michael Edward Thomas |
| New Yorker on Bridge | Jeanie Fox |
| Tugboat Captain | Robert Kerman |
| Man in Fair (uncredited) | Stan Lee |
| Lab Tour Guide (uncredited) | Una Damon |
| Cop (uncredited) | Rick Avery |
| Colonel (uncredited) | Peter Aylward |
| Crying Girl in Tram (uncredited) | Jillian Clare |
| Kyle (uncredited) | Chris Coppola |
| Spectator at School Fight (uncredited) | Jesse Heiman |
| World Unity Festival Attendant (uncredited) | Leroy Patterson |
| Mugger (uncredited) | Benny Urquidez |
| Screaming Wrestler (uncredited) | Scott L. Schwartz |
| Chef (uncredited) | Jophery C. Brown |
| Young Thug #2 (uncredited) | Brian J. Williams |
| Cop (uncredited) | Mark De Alessandro |
| Uptown Woman (uncredited) | Tia Dionne Hodge |
| Board of Directors Member (uncredited) | Loren Janes |
| Balkan's Aide (uncredited) | Andray Johnson |
| Thug on 8x10 (uncredited) | Martin Pfefferkorn |
| Street Vendor (uncredited) | Tammi Sutton |
| Mary Jane's Friend (uncredited) | Lindsay Thompson |
| Boat Light Man (uncredited) | Sean Valla |
| Pentagon Officer (uncredited) | Pete Macnamara |
| Cop (uncredited) | Al Goto |
| Cop (uncredited) | Joni Avery |
| Popcorn Thrower (uncredited) | Sam Raimi |
| Man with Bike (uncredited) | Craig 'Radio Man' Castaldo |
| Enrique | Lou Torres |
