Peroxisome proliferator-activated receptor γ (PPARγ) is a member of the nuclear receptor superfamily that acts as a key player in adipocyte differentiation, glucose metabolism, and macrophage differentiation. Osteopontin (OPN), a component of extracellular matrix, is elevated during neointimal formation in the vessel wall and is synthesized by macrophages in atherosclerotic plaques. In the present study, we investigated the molecular mechanisms regulating OPN gene expression by PPARγ in THP-1 cells, a cell line derived from human monocytic leukemia cells. Northern and Western blot analyses showed that exposure of THP-1 cells to PMA (phorbol 12-myristate 13-acetate) increases OPN mRNA and protein levels in a time-dependent manner. PMA-induced OPN expression was significantly decreased by troglitazone (Tro) and other PPARγ ligands. Transient transfection assays of the human OPN promoter/luciferase construct showed that PPARγ represses OPN promoter activity, and the PPARγ-responsive region within the OPN promoter lies between −1000 and −970 relative to the transcription start site. Site-specific mutation analysis and electrophoretic mobility shift assays indicated that a homeobox-like A/T-rich sequence between −990 and −981, which functions as a binding site for PMA-induced nuclear factors other than PPARγ, mediates the repression of OPN expression by Tro. Furthermore, concatenated A/T-rich sequences conferred the PPARγ responsiveness on the heterologous promoter. Taken together, these data suggest that PPARγ ligand inhibits OPN gene expression through the interference with the binding of nuclear factors to A/T-rich sequence in THP-1 cells.