Abstract West Nile virus (WNV)-induced neurological disease has become a public health concern. No vaccines have been approved for human use. We have previously shown that an attenuated WNV, the nonstructural(NS)4B-P38G mutant, induced stronger innate cytokine and T cell responses than wild-type WNV. Mice immunized with this mutant were all protected from subsequent lethal wild-type WNV infection. Furthermore, TLR7 and MyD88 expression is required for host protection from NS4B P38G infection. Here, we have examined innate cytokine response in THP-1 cells (human monocytic cells) and THP-1 derived macrophages following with NS4B P38G infection. We have found that NS4B P38G induced stronger type 1 IFN and pro-inflammatory cytokine responses in THP-1 cells than those infected by wild-type WNV. Interestingly, we noted that there was a higher production of pro-inflammatory cytokines, but not IFN-b in NS4B P38G infected THP-1 derived macrophages. Our results also showed an upregulation of TLR4, TLR7 and RIG-I expression in THP-1 cells following NS4B P38G infection, whereas TLR2 expression was significantly increased in mutant infected THP-1 derived macrophages. Similar as our findings in mice, NS4B P38G mutant had a very low replication rate in both types of human cells. Overall, these results suggest that NS4B P38G induced a differential innate cytokine response in human monocytic cells and macrophages, possibly via different pathogen recognition receptor signaling pathways in these cells.