Japanese encephalitis virus (JEV) is a mosquito-borne Flavivirus that causes Japanese encephalitis (JE) in eastern and southern Asia. An estimated 3 billion persons live in countries where JE is endemic. So far, no effective antiviral therapy is available except for the vaccination. There is a need for effective strategies for the development of anti-JEV agents. Antiviral agents from natural products have been extensively studied. Here, we investigated the anti-JEV activity of the recombinant Griffithsin (GRFT) which may help in the development of a new antiviral drug to combat the JEV infection. GRFT is a carbohydrate-binding protein, derived from the red alga Griffithsia sp. that has been shown to interfere with the cellular attachment and infectivity of a number of enveloped viruses, including HIV-1. We first de novo synthesized, cloned, expressed and purified the GRFT recombinantly from Escherichia coli system. Next, the antiviral activity of GRFT against JEV infection in vitro and in vivo was evaluated. Finally, the possible mechanism by which GRFT inhibits JEV infection in BHK-21 cells was investigated.