Japanese encephalitis virus (JEV) is a member of the Flaviviridae family and one of Asia’s most common causes of encephalitis. JEV is a zoonotic virus that is transmitted to humans through the bite of infected mosquitoes of theCulexspecies. While humans are dead-end hosts for the virus, domestic animals such as pigs and birds are amplification hosts. Although JEV naturally infected monkeys have been reported in Asia, the role of non-human primates (NHPs) in the JEV transmission cycle has not been intensively investigated. In this study, we demonstrated neutralizing antibodies against JEV in NHPs (Macaca fascicularis) and humans living in proximity in two provinces located in western and eastern Thailand by using Plaque Reduction Neutralization Test (PRNT). We found a 14.7% and 5.6% seropositive rate in monkeys and 43.7% and 45.2% seropositive rate in humans living in west and east Thailand, respectively. This study observed a higher seropositivity rate in the older age group in humans. The presence of JEV neutralizing antibodies in NHPs that live in proximity to humans shows the occurrence of natural JEV infection, suggesting the endemic transmission of this virus in NHPs. According to the One Health concept, regular serological studies should be conducted especially at the animal–human interface.