Japanese encephalitis (JE) developed in an unvaccinated half-bred horse kept in Tottori Prefecture, Japan. The animal showed ataxia with pyrexia and low appetite, and ultimately died. A viral strain was isolated from the cerebrum of the horse and was identified as JE virus (JEV) by RT-PCR using JEV specific primers. The isolated JEV was classified into genotype I by nucleotide sequence analysis of the viral envelope gene. We believe that this is the first report of the genotype I strain being isolated from a horse.