It was shown previously that the highly conserved vaccinia virus A35 gene is an important virulence factor in respiratory infection of mice. We show here that A35 is also required for full virulence by the intraperitoneal route of infection. A virus mutant in which the A35 gene has been removed replicated normally and elicited improved antibody, gamma interferon-secreting cell, and cytotoxic T-lymphocyte responses compared to wild-type virus, suggesting that A35 increases poxvirus virulence by immunomodulation. The enhanced immune response correlated with an improved control of viral titers in target organs after the development of the specific immune response. Finally, the A35 deletion mutant virus also provided protection from lethal challenge (1,000 50% lethal doses) equal to that of the wild-type virus. Together, these data suggest that A35 deletion viruses will make safer and more efficacious vaccines for poxviruses. In addition, the A35 deletion viruses will serve as improved platform vectors for other infectious diseases and cancer and will be superior vaccine choices for postexposure poxvirus vaccination, as they also provide improved kinetics of the immune response.