Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR -/-) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β -/- mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β -/- mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR -/- mice. The increased susceptibility of IFN-β -/- mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β -/- mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8 + T cells, we observed an altered CD4 + CD25 + FoxP3 + regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.