Smallpox, caused by variola virus, was a devastating disease in humans, but how the virus evolved a strategy to spread to tissue remains unknown. Through the use of microarrays, we identified the gene encoding the Wiskott-Aldrich syndrome protein (WASP), one of the five known WASP family members, which has been induced in the course of infection of human cells with different strains of vaccinia virus (VV) (S. Guerra, L. A. López-Fernández, A. Pascual-Montano, M. Muñoz, K. Harshman, and M. Esteban, J. Virol. 77:6493-6506, 2003; S. Guerra, L. A. López-Fernández, R. Conde, A. Pascual-Montano, K. Harshman, and M. Esteban, J. Virol. 78:5820-5834, 2004). In a mouse model, we evaluated the role of WASP in infection with VV, a close relative of variola virus. WASP−/− (KO) mice infected intranasally and intraperitoneally with VV showed reduced weight loss and mortality compared to wild-type (WT) mice. WASP expression correlated with VV replication in the ovaries but not in the liver or spleen. WT mouse macrophages express WASP but not N-WASP; after VV infection, WASP levels increase threefold. KO macrophages lack N-WASP expression and, when VV infected, are incapable of inducing actin tails and producing extracellular virus. These functions were rescued in KO macrophages after ectopic WASP expression. Overall, our findings demonstrate that WASP has a role in orthopoxvirus infections. Use of WASP proteins for virus spread via the actin tail provides a selective advantage for VV, and probably variola virus, dissemination to distant tissues.