We generated a panel of 46 monoclonal antibodies against WNV E protein, and applied a new high-throughput epitope-mapping strategy to identify a dominant epitope that was recognized by the majority of neutralizing monoclonal antibodies in DIII. This epitope was also detected by convalescent antibodies from individuals who had recovered from WNV infection without clinical consequence. Three neutralizing monoclonal antibodies protected against WNV mortality in a postexposure therapy model. One of these, E16, was humanized and confirmed as therapeutically effective in mice.