Resistin antagonizes insulin action in mouse, making it a potential therapeutic target for treating metabolic diseases such as diabetes. To better understand how mouse resistin gene (Retn) expression is restricted to fat tissue, we identified an adipocyte-specific enhancer located approximately 8.8-kb upstream of the transcription start site. This region contains a binding site for the master adipogenic regulator peroxisome proliferator-activated receptor gamma (PPARgamma), and binds endogenous PPARgamma together with its partner retinoid-X receptor alpha (RXRalpha). It also contains three binding sites for CCAAT/enhancer-binding protein (C/EBP), and is bound by endogenous C/EBPalpha and C/EBPbeta in adipocytes. Exogenous expression of PPARgamma/RXRalpha and C/EBPalpha in non-adipocyte cells synergistically drives robust expression from the enhancer. Although PPARgamma ligands repress Retn transcription in adipocytes, rosiglitazone paradoxically stimulates the enhancer activity, suggesting that the enhancer is not directly involved in negative regulation. Unlike expression of Retn in mouse, human resistin (RETN) is expressed primarily in macrophages. Interestingly, the region homologous to the mouse Retn enhancer in the human gene contains all three C/EBP elements, but is not conserved for the sequence bound by PPARgamma. Furthermore, it displays little or no binding by PPARgamma in vitro. Taken together, the data suggest that a composite enhancer binding both PPARgamma and C/EBP factors confers adipocyte-specific expression to Retn in mouse, and its absence from the human gene may explain the lack of adipocyte expression in humans.