Background：Japanese encephalitis virus (JEV), as a re-emerging virus that causes 10,000-15,000 human deathsfrom encephalitis in the world each year, has had a significant impact on public health.Pigs are the naturalreservoirs of JEV and play an important role in the amplification, dispersal and epidemiology of JEV. The nonstructural protein 3 (NS3) of JEV possesses enzymatic activities of serine protease, helicase and nucleoside 5-triphosphatase, and plays important roles in viral replication and pathogenesis.Results：We characterized the NS3 protein of a neurovirulent strain of JEV (SH-JEV01)isolated from a field-infectedpig. TheNS3 gene of the JEV SH-JEV01 strain is 1857bp in length and encodes protein of approximately 72 kDawith 99％amino acid sequence identity to that of the representative immunotype strain JaGAr 01. The NS3 proteinwas detectable 12h post-infection in a mouse neuroblastoma cell line, Neuro-2a, and was distributed inthe cytoplasm of cells infected with the SH-JEV01 strain of JEV. In the brain of mice infected with the SH-JEV01 strainof JEV, NS3 was detected in the cytoplasm of neuronal cells, including pyramidal neurons of the cerebrum, granulecells, small cells and Purkinje cells of the cerebellum.Conclusions：The NS3 protein of a neurovirulent strain of JEV isolated from a pig was characterized. It is anapproximately 72 kDa protein and distributed in the cytoplasm of infected cells. The Purkinje cell of the cerebellumis one of the target cells of JEV infection. Our data should provide some basic information for the study of the roleof NS3 in the pathogenesis of JEV and the immune response.