The nationwide introduction of a Japanese encephalitis (JE) vaccine has contributed to a reduction in the annual infection rate of JE in Japan. However, the current neutralizing antibody prevalence ratio in Japan is approximately 20% in children 3-4 years of age and in people in their forties and fifties. We herein report a man with JE who was definitively diagnosed by multi-virus real-time polymerase chain reaction employing biopsied brain tissue and serological examinations. JE should be kept in mind when a patient has severe encephalitis of unknown etiology. In order to protect the susceptible population from JE, vaccination is recommended, especially for children and middle-aged people.