The antiviral effects of nitric oxide (NO) on Japanese encephalitis virus (JEV), a member of the family Flaviviridae, were investigated in this study. In vitro, inhibition of replication of JEV in gamma interferon-activated RAW 264.7 murine macrophages was correlated to cellular NO production. When cocultured with infected murine neuroblastoma N18 cells, gamma interferon-activated RAW 264.7 cells also efficiently hindered JEV replication in contiguous bystanders, and this anti-JEV effect could be reversed by an NO synthase (NOS) inhibitor, N-monomethyl-L-arginine acetate. In vivo, the mortality rate increased as the NOS activity of JEV-infected mice was inhibited by its competitive inhibitor, N-nitro-L-arginine methyl ester. Moreover, when an organic donor, S-nitro-N-acetylpenicillamine (SNAP), was used, the NO-mediated antiviral effect was also observed in primarily JEV-infected N18, human neuronal NT-2, and BHK-21 cells, as well as in persistently JEV-infected C2-2 cells. These data reaffirm that NO has an effective and broad-spectrum antimicrobial activity against diversified intracellular pathogens. Interestingly, the antiviral effect of NO was not enhanced by treatment of N18 cells with SNAP prior to JEV infection, a measure which has been shown to greatly increase the antiviral effect of NO in infection by vesicular stomatitis virus. From biochemical analysis of the impact of NO on JEV replication in cell culture, NO was found to profoundly inhibit viral RNA synthesis, viral protein accumulation, and virus release from infected cells. The results herein thus suggest that NO may play a crucial role in the innate immunity of the host to restrict the initial stage of JEV infection in the central nervous system.