West Nile virus (WNV) causes serious neurologic disease, but no licensed vaccines are available to prevent this disease in humans. We have developed RepliVAX WN, a single-cycle flavivirus with an expected safety profile superior to other types of live-attenuated viral vaccines. In this report we describe studies examining RepliVAX WN safety, potency, and efficacy in a non-human primate model of WNV infection. A single immunization of four rhesus macaques with RepliVAX WN was safe and elicited detectable neutralizing antibody titers and IgM and IgG responses, and IgG titers were increased in two animals that received a second immunization. After challenge with WNV, three of four immunized animals were completely protected from viremia, and the remaining animal showed minimal viremia on one day. In contrast, the unvaccinated animal developed viremia that lasted six days. These results demonstrate the efficacy and safety of RepliVAX WN in this primate model of WNV infection.