Interferon regulatory factor (IRF)-3 is a master transcription factor that activates host antiviral defense programs. Although cell culture studies suggest that IRF-3 promotes antiviral control by inducing interferon (IFN)-β, near normal levels of IFN-α and IFN-β were observed in IRF-3 -/- mice after infection by several RNA and DNA viruses. Thus, the specific mechanisms by which IRF-3 modulates viral infection remain controversial. Some of this disparity could reflect direct IRF-3-dependent antiviral responses in specific cell types to control infection. To address this and determine how IRF-3 coordinates an antiviral response, we infected IRF-3-/- mice and two primary cells relevant for West Nile virus (WNV) pathogenesis, macrophages and cortical neurons. IRF-3-/- mice were uniformly vulnerable to infection and developed elevated WNV burdens in peripheral and central nervous system tissues, though peripheral IFN responses were largely normal. Whereas wild-type macrophages basally expressed key host defense molecules, including RIG-I, MDA5, ISG54, and ISG56, and restricted WNV infection, IRF-3-/- macrophages lacked basal expression of these host defense genes and supported increased WNV infection and IFN-α and IFN-β production. In contrast, wild-type cortical neurons were highly permissive to WNV and did not basally express RIG-I, MDA5, ISG54, and ISG56. IRF-3-/- neurons lacked induction of host defense genes and had blunted IFN-α and IFN-β production, yet exhibited only modestly increased viral titers. Collectively, our data suggest that cell-specific IRF-3 responses protect against WNV infection through both IFN-dependent and -independent programs.