West Nile Virus (WNV) is a mosquito-transmitted flavivirus which causes encephalitis especially in elderly and immunocompromised individuals. Previous studies have suggested the protective role of the Toll-like receptor 3 (TLR3) pathway against WNV entry into the brain, while the WNV non-structural protein 1 (NS1) interferes with the TLR3 signaling pathway, besides being a component of viral genome replication machinery. In this study, we investigated whether immunization with NS1 could protect against WNV neuroinvasion in the context of TLR3 deficiency. We immunized mice with either an intact or deleted TLR3 system (TLR3KO) with WNV envelope glycoprotein (gE) protein, NS1, or a combination of gE and NS1. Immunization with gE or gE/NS1, but not with NS1 alone, induced WNV neutralizing antibodies and protected against WNV brain invasion and inflammation. The presence of intact TLR3 signaling had no apparent effect on WNV brain invasion. However, mock-immunized TLR3KO mice had higher inflammatory cell invasion upon WNV brain infection than NS1-immunized TLR3KO mice and wild type mice. Thus, immunization against NS1 may reduce brain inflammation in a context of TLR3 signaling deficiency.