Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-κB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-κB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-κB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines.