Transforming growth factor-β1 (TGF- β1) and thrombospondin-1 (TSP-1) are hypoxia-responsive mitogens that promote vascular smooth muscle cell (SMC) proliferation, a critical event in the pathogenesis of pulmonary hypertension (PH). We previously demonstrated that hypoxia-induced human pulmonary artery smooth muscle (HPASMC) cell proliferation and expression of the NADPH oxidase subunit, Nox4, were attenuated by the peroxisome proliferator-activated receptor γ (PPARγ) agonist, rosiglitazone. The current study examines the hypothesis that rosiglitazone regulates Nox4 expression and HPASMC proliferation by attenuating TSP-1 signaling. Selected HPASMC were exposed to normoxic or hypoxic (1% O2) environments or TSP-1 (0-1 μg/ml) for 72 hours ± administration of rosiglitazone (10 μM). Cellular proliferation, Nox4, TSP-1, and TGF-β1 expression and reactive oxygen species generation were measured. Mice exposed to hypoxia (10% O2) for three weeks were treated with rosiglitazone (10 mg/kg/day) for the final 10 days, and lung TSP-1 expression was examined. Hypoxia increased TSP-1 and TGF-P1 expression and HPASMC proliferation, and neutralizing antibodies to TSP-1 or TGF-β1 attenuated proliferation. Rosiglitazone attenuated hypoxia-induced HPASMC proliferation and increases in mouse lung and HPASMC TSP-1 expression, but failed to reduce increases in TGF-β1 expression or Nox4 expression and activity caused by direct TSP-1 stimulation. Transfecting HPASMC with siRNA to Nox4 attenuated hypoxia- ∗∗∗or TSP-1-stimulated HPASMC proliferation. These findings provide novel evidence that TSP-1-mediated Nox4 expression plays a critical role in hypoxia-induced HPASMC proliferation. PPARγ activation with exogenous ligands attenuates TSP-1 expression to reduce Nox4 expression. These results clarify mechanisms of hypoxia-induced SMC proliferation and suggest additional pathways by which PPARγ agonists may regulate critical steps in the pathobiology of PH.