We examined whether live attenuated Japanese encephalitis (JE) vaccine is effective in preventing West Nile virus (WNV) infection in the People's Republic of China. Three groups were recruited into the study: patients with Japanese encephalitis (JE), healthy controls vaccinated with live attenuated 2 SA14-14-vaccine against JE virus (JEV), and unvaccinated healthy controls. Serum samples were collected and screened for IgG antibodies against JEV by an indirect immunofluorescence assay. Positive samples were then analyzed for levels of antibodies against JEV and neutralizing antibodies against West Nile virus (WNV) by a plaque-reduction neutralization test (PRNT). Although most persons had medium to high levels of JEV-reactive IgG and neutralizing antibodies, only 2 of the 82 unvaccinated control samples were positive for the WNV-reactive antibodies. These findings suggest that previous JEV infection or vaccination did not induce adequate levels of WNV-reactive antibodies in the population studied. However, how these persons would respond to a secondary flavivirus infection and whether their prior experience with wild-type or attenuated JE vaccine will provide some cross-protection against WNV disease still warrants further investigation.