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- Bruce Greenwood as Batman
- Jensen Ackles as Red Hood
- John DiMaggio as The Joker
- Neil Patrick Harris as Nightwing
- Jason Isaacs as Ra's al Ghul
- Wade Williams as Black Mask
- Gary Cole as Bobo, James Gordon, Shot
- Kelly Hu as Ms. Li
- Jim Piddock as Alfred Pennyworth
- Carlos Alazraqui as Chi Chi and Baton
- Robert Clotworthy as Leon
- Phil LaMarr as Rick
- Vincent Martella as Robin
- Alexander Martella as Robin
- Kevin Michael Richardson as Tyler Bramford
- Dwight Schultz as Freddie and Yurp
- Fred Tatasciore as Amazo
- Bruce Timm as The Riddler