Previous studies have demonstrated that KOR activation results in decreased susceptibility to infection by HIV-1 in human PBMCs. In the present studies, we have found this effect is, in part, a result of down-regulation of the major HIV-1 coreceptor, CXCR4. Using a combination of biochemical approaches, our results show that CXCR4 protein and mRNA levels were reduced significantly following KOR activation. We evaluated the nature of the signaling pathway(s), which were induced by KOR activation, using transcription factor-binding array analysis and comparing extracts from control and KOR-activated cells. We determined that the IRFs and STATs were induced following KOR activation, and these events were important for the inhibition of CXCR4 expression. Using chemical inhibitors and siRNA constructs, we determined that JAK2, STAT3, and IRF2 were critical members of this signal transduction pathway. Immediately following KOR activation, JAK2 was phosphorylated, and this was required for the phosphorylation/activation of STAT3. Moreover, IRF2 mRNA and protein expression were also up-regulated, and further studies using ChIP analysis showed that IRF2 was induced to bind in vivo to the CXCR4 promoter. This is the first report detailing the initiation of a KOR-induced JAK2/STAT3 and IRF2 signaling cascade, and these pathways result in substantial down-regulation of CXCR4 expression. The capacity of KOR to down-regulate CXCR4 expression may provide a strategy for the development of novel therapeutics for the inhibition of HIV replication.