The potential use of smallpox as an agent of bioterrorism has renewed interest in the development of a modern vaccine capable of replacing the standard Dryvax^® vaccine. Vaccinia virus (ACAM2000), clonally isolated from Dryvax^® and manufactured in cell culture, was tested for immunogenicity and protective activity in a non-human primate model. Cynomolgus monkeys vaccinated with ACAM2000, Dryvax^®, or ACAM2000 diluent (control) were challenged 2 months post-vaccination with a lethal, intravenous dose of monkeypox virus. ACAM2000 proved immunogenic and efficacious in protecting against lethal monkeypox challenge, as evident from a lack of post-challenge viral replication, and the absence of any significant clinical signs attributable to monkeypox infection. This protection correlated (with) neutralizing antibody titers equivalent to those generated in the Dryvax^® group post-vaccination, as well as a similar significant increase in the presence of neutralizing antibodies post-challenge. Control animals showed no signs of vaccine-induced seroconversion, displayed post-challenge tissue-associated viral replication and viremia, and developed severe monkeypox-specific clinical symptoms. The protective efficacy of ACAM2000 was found to be equivalent to the currently approved vaccine, Dryvax^®.