Previous mutational analyses of naturally occurring West Nile virus (WNV) strains and engineered mutant WNV strains have identified locations in the viral genome that can have profound phenotypic effect on viral infectivity, temperature sensitivity and neuroinvasiveness. We chose six mutant WNV strains to evaluate for vector competence in the natural WNV vector Culex tarsalis, two of which contain multiple ablations of glycosylation sites in the envelope and NS1 proteins; three of which contain mutations in the NS4B protein and an attenuated natural bird isolate (Bird 1153) harbouring an NS4B mutation. Despite vertebrate attenuation, all NS4B mutant viruses displayed enhanced vector competence by Cx. tarsalis. Non-glycosylated mutant viruses displayed decreased vector competence in Cx. tarsalis mosquitoes, particularly when all three NS1 glycosylation sites were abolished. These results indicate the importance of both the NS4B protein and NS1 glycosylation in the transmission of WNV by a significant mosquito vector.