The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a member of the nuclear hormone receptor superfamily. Ligand activation of PPARgamma is associated with differentiation and inhibition of proliferation in the normal and malignant cells. Herein, we studied the effects of PPARgamma and the PPARgamma agonists thiazolidinediones (TZDs) on the insulin receptor (IR), a cell membrane tyrosine kinase receptor protein, whose role is of paramount importance in mediating the metabolic and growth-promoting effects of the peptide hormone insulin. Overexpression of the PPARgamma1 in human hepatocellular (HepG2) cells was associated with decreased IR gene transcription and protein expression levels, and these reductions were more evident in the presence of TZDs. Since no PPARgamma response elements were identified on the IR promoter, we postulated that PPARgamma adversely affects the IR gene transcription by perturbing the assembly and stability of the transcriptionally active multiprotein-DNA complex identified previously, which includes the high-mobility group A1 protein, the ubiquitously expressed transcription factor (Sp1), the CAAT enhancer-binding protein (C/EBPbeta), and, in some cell lines, the developmentally regulated activator protein-2 (AP-2) transcription factor. Using glutathione S-transferase pull-down assays combined with electrophoretic mobility shift assay and chromatin immunoprecipitation, we demonstrated that by interacting with Sp1, C/EBPbeta, and AP-2, PPARgamma can prevent Sp1/AP-2 protein-protein association and inhibit binding of Sp1 and C/EBPbeta to DNA, thus reducing IR gene transcription. Our results demonstrate that IR is a new target gene of PPARgamma, and support a potential use of TZDs as anti-proliferative agents in selected neoplastic tissues overexpressing IRs.