Tumor necrosis factor alpha (TNFalpha) expression is a key mediator of ethanol-induced liver disease. Increased lipopolysaccharide (LPS)-stimulated TNFalpha expression in macrophages after chronic ethanol feeding is associated with a stabilization of TNFalpha mRNA (Kishore, R., McMullen, M. R., and Nagy, L. E. (2001) J. Biol. Chem. 276, 41930-41937). Here we show that the 3'-UTR of murine TNFalpha mRNA was sufficient to mediate increased LPS-stimulated expression of a luciferase reporter in RAW 264.7 macrophages after chronic ethanol exposure. Further, we show that HuR, a nuclear/cytoplasmic shuttling protein, which binds to TNFalpha mRNA, is required for increased expression of TNFalpha after chronic ethanol. In Kupffer cells, HuR was primarily localized to the nucleus and then translocated to the cytosol in response to LPS in both pair- and ethanol-fed rats. After chronic ethanol feeding, HuR quantity in the cytosol was greater, both at baseline and in response to LPS, compared with pair-fed controls. Using RNA gel shift assays, we found that LPS treatment increased HuR binding to the 65-nucleotide A + U-rich element of the TNFalpha 3'-UTR by 2-fold over baseline in Kupffer cells from pair-fed rats. After chronic ethanol feeding, HuR binding to the TNFalpha A + U-rich element was increased by more than 5-fold at baseline and in response to LPS, compared with pair-fed controls. Down-regulation of HuR expression by RNA interference prevented the chronic ethanol-induced increase in expression of luciferase reporters containing the TNFalpha 3'-UTR. Taken together, these data demonstrate that increased binding of HuR to the TNFalpha 3'-UTR contributes to increased LPS-stimulated TNFalpha expression in macrophages after chronic ethanol exposure.