Japanese encephalitis virus (JEV) remains a leading cause of viral encephalitis worldwide. Although JEV-specific antibodies have been described, an assessment of their ability to neutralize multiple genotypes of JEV has been limited. Here, we describe the development of a panel of mouse and human neutralizing monoclonal antibodies (MAbs) that inhibit infection in cell culture of four different JEV genotypes tested. Mechanism-of-action studies showed that many of these MAbs inhibited infection at a postattachment step, including blockade of virus fusion. Mapping studies using site-directed mutagenesis and hydrogen-deuterium exchange with mass spectrometry revealed that the lateral ridge on domain III of the envelope protein was a primary recognition epitope for our panel of strongly neutralizing MAbs. Therapeutic studies in mice demonstrated protection against lethality caused by genotype I and III strains when MAbs were administered as a single dose even 5 days after infection. This information may inform the development of vaccines and therapeutic antibodies as emerging strains and genotypic shifts become more prevalent.IMPORTANCE Although Japanese encephalitis virus (JEV) is a vaccine-preventable cause of viral encephalitis, the inactivated and live attenuated platforms available are derived from strains belonging to a single genotype (GIII) due to its historical prevalence in areas of JEV epidemics. Related to this, studies with vaccines and antibodies have focused on assessing the in vitro and in vivo protective responses to homologous or heterologous GIII strains. An epidemiological shift in JEV genotype distribution warrants the induction of broadly neutralizing antibody responses that inhibit infection of multiple JEV genotypes. Here, we generated a panel of mouse and human neutralizing monoclonal antibodies and evaluated their inhibitory activity, epitope location, and capacity for protection against multiple JEV genotypes in mice.