Japanese encephalitis virus (JEV) causes fatal diseases in equines as well as humans. In Japan, racehorses are vaccinated with inactivated JE vaccine every year and no equine JE cases have been reported since 1986. However, the current reduction in JEV activity in nature has raised an argument against the requirement of continuous vaccination. Here, we studied natural infection rates in racehorses to address the issue. To identify naturally-infected individuals from vaccinated populations, we used an immunostaining method for detecting antibodies to JEV nonstructural 1 (NS1) protein. A total of 779 horses in eight nationwide locations showed NS1 antibody prevalences ranging from 15 to 73%. NS1 antibody prevalences among 2-year-old individuals that had spent one epizootic season in a particular location, therefore representing annual infection rates, were 15-67%. Individuals aged >/=3 years showed higher NS1 antibody titers than the 2-year-olds, suggesting that NS1 antibody levels were boosted by repeated exposures to JEV antigen over >/=2 epizootic seasons. These results indicate that horses in Japan are exposed to natural JEV infections, confirming the need for continuous vaccination for protecting horses from JE.