BACKGROUND: Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, mainly among adults. JCV is widely distributed in North America and the number of JCV cases in the U.S. has increased in recent years. Therefore, the central nervous system disease caused by JCV can be considered a potentially re-emerging viral disease. However, the seroprevalence of JCV is unknown in Japan. The purpose of this study is to evaluate the seroprevalence of JCV in the Japanese population. METHODS: We used an IgG enzyme-linked immunosorbent assay (IgG-ELISA) with JCV-infected cell-lysates and/or a neutralizing (NT) antibody assay. The cut-off value of IgG-ELISA was determined using IgG-ELISA to analyze serum specimens from 37 healthy Japanese donors. IgG-ELISA was validated by assessing its sensitivity and specificity, using 38 human serum samples previously tested for the presence or absence of antibodies against JCV and snowshoe hare virus (SSHV), in an in-house NT antibody assay conducted by the Public Health Agency of Canada. The seroepidemiological study was performed using IgG-ELISA and NT antibody assay to analyze 246 human serum samples from the serum bank of the National Institute of Infectious Diseases (NIID) in Japan. RESULTS: The cut-off value of IgG-ELISA was determined at 0.20, based on the mean (- 0.075) and standard deviation (0.092) values using Japanese donors' sera. The sensitivity and the specificity of IgG-ELISA determined using 25 JCV-positive and 4 JCV-negative serum samples were 96 and 100%, respectively. Analysis of the 246 Japanese serum samples revealed that no specimen showed a higher value than the cut-off value of IgG-ELISA, and no sample tested positive by the NT antibody assay. CONCLUSIONS: Our results showed that JCV is not circulating significantly in Japan. To the best of our knowledge, this is the first report to demonstrate the seroprevalence of JCV in the general population in Japan.