•MyD88 and Trif regulate CNS expression of anti-viral cytokines and affect survival in WNV infection.•WNV-infected MyD88/Trif−/− had early viremia peaks and encephalitis compared to WT mice.•WNV in MyD88/Trif−/− increased gliosis and exacerbated inflammatory influx.•TLR signaling pathways prevent WNV-induced lethal encephalitis at early stages of infection.Toll-like receptors (TLRs) are known to be activated in Central Nervous System (CNS) viral infections and are recognized to be a critical component in innate immunity. Several reports state a role for particular TLRs in various CNS viral infections. However, excessive TLR activation was previously reported by us in correlation with a pathogenic, rather than a protective, outcome, in a model of SIV encephalitis. Here we aimed at understanding the impact of TLR-mediated pathways by evaluating the early course of pathogenesis in the total absence of TLR signaling during CNS viral infections. We utilized a mouse model of sublethal West Nile virus (WNV) infection. WNV is an emerging neurotropic flavivirus, and a significant global cause of viral encephalitis. The virus was peripherally injected into animals that simultaneously lacked two key adapter molecules of TLR signaling, MyD88 and TRIF. On day 2 pi (post infection), MyD88/Trif−/− mice showed an increased susceptibility to WNV infection, and revealed an impairment in innate immune cytokines, when compared to wild type mice (WT). By day 6 pi, there was an increase in viral burden and robust expression of inflammatory cytokines as well as higher cell infiltration into the CNS in MyD88/Trif−/−, when compared to infected WT. A drastic increase in microglia activation, astrogliosis, and inflammatory trafficking were also observed on day 6 pi in MyD88/Trif−/−. Our observations show a protective role for TLR signaling pathways in preventing lethal encephalitis at early stages of WNV infection.