The neutralization of Japanese encephalitis virus (JEV) was studied using JEV-specific neutralizing (NT) monoclonal antibody (MAb) 503 that recognizes the envelope glycoprotein. Analysis using radiolabeled JEV and observations by confocal laser microscopy and electron microscopy indicated that the NT and protection activities of MAb 503 did not result from the prevention of the first step of JEV infection, binding of virus to the cell surface. Treatment with MAb 503 strongly inhibited JEV-induced cell fusion and internalization of JEV into the cells, and resulted in enhanced release of JEV-RNA from the cells. These observations suggested that the NT activity of MAb 503 is involved in the later steps of JEV infection.