In this study, we establish a major role for RLR signaling in protection from WNV pathogenesis, and demonstrate that IPS-1 is critical for the control of WNV infection in vivo. Our studies indicate that IPS-1-dependent RLR signaling functions to establish balanced, effective, and protective innate and adaptive immune responses, and that IPS-1 links adaptive immune regulation with the innate immunity triggered by RLR signaling during WNV infection. In the absence of IPS-1 in vitro, innate immune defense programs of myeloid DCs and macrophages were ablated or severely attenuated. Moreover, in vivo analysis of infected IPS-1 2/ 2 mice showed altered IgG and IgM antibody responses with diminished virus neutralization activity. The inflammatory response to WNV infection is regulated by IPS-1-dependent processes such that a deficiency of IPS-1 resulted in elevated proinflammatory cytokine and chemokines and increased numbers of inflammatory DCs, antigen-specific T cells, natural killer cells, and neutrophils in lymphoid organs, and activated macrophage/ microglial cells within the CNS. The dysregulated inflammatory response to WNV infection in IPS-1 2/2 mice was associated with a reduction in the numbers of T reg cells and their failure to expand during acute infection. These observations demonstrate the critical role of IPS-1 in mediating RLR signaling of innate antiviral immunity against WNV infection, and reveal novel features of IPS-1 function in regulating immune homeostasis, inflammation, and adaptive immunity to infection.