The important roles of a nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPARgamma activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPARgamma activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPARgamma. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPARgamma-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPARgamma.