ABSTRACT West Nile virus (WNV) is a mosquito-borne flavivirus that is neurotropic in humans, birds, and other animals. While adaptive immunity plays an important role in preventing WNV spread to the central nervous system (CNS), little is known about how alpha/beta interferon (IFN-α/β) protects against peripheral and CNS infection. In this study, we examine the virulence and tropism of WNV in IFN-α/β receptor-deficient (IFN- α/βR −/− ) mice and primary neuronal cultures. IFN-α/βR −/− mice were acutely susceptible to WNV infection through subcutaneous inoculation, with 100% mortality and a mean time to death (MTD) of 4.6 ± 0.7 and 3.8± 0.5 days after infection with 10 0 and 10 2 PFU, respectively. In contrast, congenic wild-type 129Sv/Ev mice infected with 10 2 PFU showed 62% mortality and a MTD of 11.9 ± 1.9 days. IFN-α/βR −/− mice developed high viral loads by day 3 after infection in nearly all tissues assayed, including many that were not infected in wild-type mice. IFN-α/βR −/− mice also demonstrated altered cellular tropism, with increased infection in macrophages, B cells, and T cells in the spleen. Additionally, treatment of primary wild-type neurons in vitro with IFN-β either before or after infection increased neuronal survival independent of its effect on WNV replication. Collectively, our data suggest that IFN-α/β controls WNV infection by restricting tropism and viral burden and by preventing death of infected neurons.