- Jason Alexander
- Narrator
- David Arquette
- Eddie
- Colleen Ballinger
- Janet Weiss
- Ben Barnes
- Brad Majors
- Lance Bass
- Rocky
- Rachel Bloom
- Janet Weiss
- Barry Bostwick
- Brad Majors
- Nell Campbell
- Columbia
- Kalen Chase
- Riff Raff
- Garrett Clayton
- Brad Majors (Musical Performance)
- Tim Curry
- Dr. Frank-N-Furter - A Scientist
- Rosario Dawson
- Magenta
- The Dresden Dolls
- Self
- Shayne Eastin
- Lips
- Jason George
- Dr. Everett V. Scott
- Eiza González
- Janet Weiss
- Frankie Grande
- Dr. Everett V. Scott
- Seth Green
- Riff Raff
- Mike McCready
- Self
- Karen Olivo
- Self
- Peppermint
- Dr. Frank-N-Furter
- Graham Phillips
- Brad Majors
- Leah Elizabeth Rucinski
- Columbia
- Keala Settle
- Self
- Patrick Stump
- Self
- Jenna Ushkowitz
- Janet Weiss
- Wilmer Valderrama
- Brad Majors
- Bob Weir
- Self
- Marissa Jaret Winokur
- Janet Weiss
