Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase ribosomal-inactivating protein isolated from Phytolacca americana. The antiviral activity of PAP has been described in several viruses. This study was to investigate the antiviral activity of PAP against Japanese encephalitis virus (JEV) infection in vitro and in vivo. Antiviral activity of PAP against JEV infection was evaluated in vitro using plaque forming assay, qRT-PCR and Western blot analysis. In vitro results showed that PAP inhibited replication of JEV in a dose-dependent manner with 50% inhibitory concentration (IC(50)) of 300 ng/ml (23.1 nM). Depurination assay suggested that the antiviral activity of PAP against JEV infection might be partially due to depurination of JEV genomic RNA. In vivo studies showed that PAP (1.0mg/kg) administered intraperitoneally decreased infection in mice challenged with a lethal dose of JEV, presenting a survival of 87.5% or 85.7% when administered pre-infection or post-infection. Collectively, our studies demonstrated that PAP possesses antiviral activity against JEV infection in vitro and in vivo, providing evidences for further development of PAP as an antiviral agent against JEV infection.