Resistin, also known as Adipocyte Secreted Factor (ADSF) and Found in Inflammatory Zone 3 (FIZZ3), is a mouse protein with potential roles in insulin resistance and adipocyte differentiation. The resistin gene is expressed almost exclusively in adipocytes. Here we show that a proximal 264-base pair fragment of the mouse resistin promoter is sufficient for expression in adipocytes. Ectopic expression of the adipogenic transcription factor CCAAT/enhancer-binding protein (C/EBPalpha) was sufficient for expression in non-adipogenic cells. C/EBPalpha binds specifically to a site that is essential for expression of the resistin promoter. Chromatin immunoprecipitation studies of the endogenous gene demonstrated adipocyte-specific association of C/EBPalpha with the proximal resistin promoter in adipocytes but not preadipocytes. C/EBPalpha binding was associated with the recruitment of coactivators p300 and CREB-binding protein and a dramatic increase in histone acetylation in the vicinity of the resistin promoter. The antidiabetic thiazolidinedione (TZD) drug rosiglitazone reduced resistin expression with an ED(50) similar to its K(d) for binding to peroxisome proliferator activated receptor gamma (PPARgamma). Other TZD- and non-TZD PPARgamma ligands also down-regulated resistin expression. However, no functional PPARgamma binding site was found within 6.2 kb of the transcriptional start site, suggesting that if PPARgamma is involved, it is either acting at a long distance from the start site, in an intron, or indirectly. Nevertheless, rosiglitazone treatment selectively decreased histone acetylation at the resistin promoter without a change in occupation by C/EBPalpha, CREB-binding protein, or p300. Thus, adipocyte specificity of resistin gene expression is because of C/EBPalpha binding, leading to the recruitment of transcriptional coactivators and histone acetylation that is characteristic of an active chromatin environment. TZD reduces resistin gene expression at least in part by reducing histone acetylation associated with the binding of C/EBPalpha in mature adipocytes.