Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFalpha production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFalpha induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFalpha production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFalpha regulation was indicated by decreased TNFalpha levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFalpha production after miR-155 overexpression, respectively. We found that miR-155 affected TNFalpha mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFalpha mRNA half-life. Using the NF-kappaB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-kappaB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-kappaB and the increased miR-155 contributes to alcohol-induced elevation in TNFalpha production via increased mRNA stability.