The seemingly invincible Spider-Man goes up against an all-new crop of villains—including the shape-shifting Sandman. While Spider-Man’s superpowers are altered by an alien organism, his alter ego, Peter Parker, deals with nemesis Eddie Brock and also gets caught up in a love triangle.
| Spider-Man / Peter Parker | Tobey Maguire |
| Mary Jane Watson | Kirsten Dunst |
| New Goblin / Harry Osborn | James Franco |
| Sandman / Flint Marko | Thomas Haden Church |
| Venom / Eddie Brock | Topher Grace |
| Gwen Stacy | Bryce Dallas Howard |
| May Parker | Rosemary Harris |
| J. Jonah Jameson | J.K. Simmons |
| Captain Stacy | James Cromwell |
| Dr. Curt Connors | Dylan Baker |
| Emma Marko | Theresa Russell |
| Joseph 'Robbie' Robertson | Bill Nunn |
| Maître D' | Bruce Campbell |
| Miss Brant | Elizabeth Banks |
| Hoffman | Ted Raimi |
| Penny Marko | Perla Haney-Jardine |
| Green Goblin / Norman Osborn | Willem Dafoe |
| Ben Parker | Cliff Robertson |
| Mr. Ditkovitch | Ilya Baskin |
| Ursula | Mageina Tovah |
| Houseman | John Paxton |
| Mrs. Stacy | Becky Ann Baker |
| Man in Times Square | Stan Lee |
| Dennis Carradine / Carjacker | Michael Papajohn |
| Flash Thompson | Joe Manganiello |
| Anchorman | Hal Fishman |
| Jennifer Dugan | Lucy Gordon |
| Photographer | Steve Valentine |
| Play Director | Tim Maculan |
| Play Producer | Marc Vann |
| Jazz Club Manager | Joe Bays |
| Precinct Detective | Gregg Daniel |
| Emergency Room Doctor | Rogelio T. Ramos |
| Crane Operator | Timothy Patrick Quill |
| Kid in Times Square | Menachem Mendel Boymelgreen |
| Kid in Times Square | Nasir Stewart |
| Kid in Times Square | Austin Hendrickson |
| Kid in Times Square | Taylor Hemhauser |
| Woman Outside Theater | Kathryn Bryding |
| Police Detective | Joe Virzi |
| New Jersey State Policeman | Bill E. Rogers |
| New Jersey State Policeman | Michael Alexander |
| Test Site Technician | April Parker Jones |
| Test Site Technician | Edward Padilla |
| Test Site Technician | Robert Curtis Brown |
| Test Site Technician | Paul Terrell Clayton |
| Test Site Technician | Carolyn Neff |
| Test Site Technician | Christina Cindrich |
| ICU Nurse | Sonya Maddox |
| Crane Disaster Radio Policeman | Andre B. Blake |
| Cop at Crane Disaster | Derrick 'Phoenix' Thomas |
| Mary Jane's Replacement | Jessi Collins |
| Boy at Key to the City Ceremony | Michael McLaughlin |
| Councilwoman | Anne Gartlan |
| Policeman at Sand Truck | Emilio Rivera |
| Policeman at Sand Truck | Keith Woulard |
| Armored Car Driver | Reynaldo Gallegos |
| Newstand Patron | Jim Coope |
| Newsstand Patron | Dean Edwards |
| Newsstand Patron | Margaret Laney |
| Congratulatory Woman at Daily Bugle | Toni Wynne |
| Coffee Shop Waitress | Aimee Miles |
| Jazz Club Waitress | Tanya Bond |
| Jazz Club Bouncer | Mark Kubr |
| Girl With Camera | Emma Raimi |
| Boy at the Final Battle | Lorne Raimi |
| Boy at the Final Battle | Henry Raimi |
| Girl at the Final Battle | Samantha Ressler |
| Jazz Club Musician | Alan Cohn |
| Jazz Club Musician | Dan Callahan |
| Jazz Club Musician | Daniel Cummings |
| Jazz Club Musician | Ron King |
| Jazz Club Musician | Carol Chaikin |
| Jazz Club Musician | Vance Hammond |
| NYPD Officer (uncredited) | Frank Anello |
| Model (uncredited) | Anya Avaeva |
| Bucket Boy (uncredited) | David Backus |
| Photoshoot Client (uncredited) | Tony Besson |
| Photographer (uncredited) | Michael Ciesla |
| Jazz Club Waitress (uncredited) | Irina Costa |
| Theater Patron (uncredited) | John Crann |
| Jazz Club Girl (uncredited) | Crystal Marie Denha |
| Columbia Grad Student (uncredited) | Amy V. Dewhurst |
| Pedestrian (uncredited) | Keith Fausnaught |
| Automobile Driver (uncredited) | Logan Fry |
| News Spectator (uncredited) | Tony Galtieri |
| News Reporter (uncredited) | Brian Hopson |
| Jazz Club Bouncer (uncredited) | Andrew James Jones |
| Girl at Key Ceremony (uncredited) | Natalie Jones |
| Student (uncredited) | Christopher Jude |
| ER Nurse (uncredited) | Brittany Krall |
| Woman in Court (uncredited) | Pierangeli Llinas |
| British Publicist (uncredited) | Bernadette Lords |
| Girl Screaming in Camaro (uncredited) | Natalie McNeil |
| Oscorp Receptionist (uncredited) | Martha Millan |
| Girl in Times Square (uncredited) | Michele-Nanette Miller |
| Robbie's Assistant (uncredited) | Claudia Katz Minnick |
| Beautiful Girl (uncredited) | Jen Oda |
| Beautiful Woman (uncredited) | Anjelia Pelay |
| Police Officer (uncredited) | Nick Poltoranin |
| Beautiful Woman (uncredited) | Vanessa Reseland |
| Beautiful Woman (uncredited) | La Rivers |
| Businesswoman (uncredited) | Bria Roberts |
| Sandman Victim (uncredited) | Vanessa Ross |
| Bad Girl (uncredited) | Brenna Roth |
| City Hall Cheerer (uncredited) | Shade Rupe |
| Café Girl (uncredited) | Abbey Skinner |
| Jazz Club Guest (uncredited) | Daniel Shafer |
| Jazz Club Patron (uncredited) | Kristin Somo |
| Jazz Club Waitress (uncredited) | Jennifer Sparks |
| NY Firefighter (uncredited) | Christopher Stadulis |
| Model (uncredited) | Aija Terauda |
| Beautiful Woman (uncredited) | Evelyn Vaccaro |
| Bar Patron #5 (uncredited) | Nick Vlassopoulos |
| Model (uncredited) | Sincerely A. Ward |
| ER Nurse (uncredited) | Jennifer Weston |
| Spectator (uncredited) | Ray Wineteer |
| Beautiful Woman (uncredited) | Emily Eckes |
| Radioman (uncredited) | Craig 'Radio Man' Castaldo |
