Abstract Japanese encephalitis virus (JEV) causes serious zoonosis in South Asia, Southeast Asia and other areas. Pigs are an important reservoir for this virus in nature. The treatment of JEV infection in pigs is important for controlling the prevalence of JEV in humans and economic losses in pig farming. In this study, we selected a high activity porcine alpha-interferon to inhibit JEV replication in porcine kidney cell lines (PK-15). Alpha interferon exhibited high antiviral activity against JEV; the expression of three interferon-stimulated genes (ISGs), ISG15, Mx2 and OAS L, increased significantly after interferon treatment. Furthermore, we verified the anti-JEV effect of these ISGs by RNAi and overexpression. Mx2 and OAS L exhibited strong anti-JEV effects in PK-15 cells. Based on these novel results, alpha interferon (IFN-α) should be considered to be a potential drug against JEV in pigs.