West Nile virus (WNV) is an emerging pathogen that is now the leading cause of mosquito-borne and epidemic encephalitis in the United States. In humans, a small percentage of infected individuals develop severe neuroinvasive disease, with the greatest relative risk being in the elderly and immunocompromised, two populations that are difficult to immunize effectively with vaccines. While inactivated and subunit-based veterinary vaccines against WNV exist, currently there is no vaccine or therapy available to prevent or treat human disease. Here, we describe the generation and preclinical efficacy of a hydrogen peroxide (H(2)O(2))-inactivated WNV Kunjin strain (WNV-KUNV) vaccine as a candidate for further development. Both young and aged mice vaccinated with H(2)O(2)-inactivated WNV-KUNV produced robust adaptive B and T cell immune responses and were protected against stringent and lethal intracranial challenge with a heterologous virulent North American WNV strain. Our studies suggest that the H(2)O(2)-inactivated WNV-KUNV vaccine is safe and immunogenic and may be suitable for protection against WNV infection in vulnerable populations.