West Nile virus (WNV) causes severe central nervous system (CNS) infection primarily in humans who are immunocompromised or elderly. In this study, we addressed the mechanism by which the immune system limits dissemination of WNV infection by infecting wild-type and immunodeficient inbred C57BL/6J mice with a low-passage WNV isolate from the recent epidemic in New York state. Wild-type mice replicated virus extraneuronally in the draining lymph nodes and spleen during the first 4 days of infection. Subsequently, virus spread to the spinal cord and the brain at virtually the same time. Congenic mice that were genetically deficient in B cells and antibody (μMT mice) developed increased CNS viral burdens and were vulnerable to lethal infection at low doses of virus. Notably, a ∼500-fold difference in serum viral load was detected in μMT mice as early as 4 days after infection, a point in the infection when low levels of neutralizing immunoglobulin M antibody were detected in wild-type mice. Passive transfer of heat-inactivated serum from infected and immune wild-type mice protected μMT mice against morbidity and mortality. We conclude that antibodies and B cells play a critical early role in the defense against disseminated infection by WNV.