The peripheral innate immune response to WNV is crucial for control of virus spread to the brain. Therefore, transcriptomes involved in the innate immune response against WNV were investigated in a model organism of rabbit peripheral blood mononuclear leukocytes (PBLs). PBLs were challenged with WNV_NSW2011 in vitro and mRNA expressions were quantified at 2h, 6h, 12h and 24h post challenge (pc) using qRT-PCR. Compared to mock infected PBL, WNV infected PBLs expressed high levels of IFNα at 6h pc. IFNγ expression increased markedly, peaking at 12h pc with a 4-fold increase. IL6, 12 & 22 expression peaked at 12h pc; while CXCL10 and PTX3 expression were higher at 2h pc and then declined. TLR1, 2, 3, 4, 6 & 10 were up-regulated at 2h pc with highest expression seen for TLR3, 4 & 6. TLRs-associated downstream genes (MyD88, STAT1, TRAF3, IRF7 & IRF9) were up-regulated between 6-24h pc. Higher expression of TRAF3, STAT1, IRF7 & IRF9 suggest the activation of TLR signaling pathways in order to detect and clear the virus. Higher expression of TNFα, HO1, iNOS, Caspase3 & Caspase9 transcripts suggest the involvement of oxidative stress and apoptosis pathway in WNV infected rabbit PBLs. The expression dynamics of selected genes were validated in PBLs from rabbits experimentally infected with WNV in vivo. Higher expression of IFNα, IFNβ, IFNγ, TNFα, IL6, IL22, PTX3, TLR3 & TLR4, IRF7 & IRF9, STST1, TRAF3, Caspase3 & Caspase9 were found in PBL from WNV infected rabbits compared to PBLs from un-infected control rabbits, and coincided with expression patterns of these genes in vitro. WNV_NSW2011 gene expression increased over time in PBLs challenged in vitro but remained undetected in PBLs from in vivo infected and control rabbits. This study highlights the array of cytokines and TLRs involved in the host innate immune response to WNV.