Hypoxia causes pulmonary arterial hypertension (PAH) by stimulating human pulmonary artery smooth muscle cell (HPASMC) proliferation. We previously demonstrated that the NADPH oxidase homologue NOX4 mediates HPASMC proliferation by transforming growth factor- 1 (TGF-1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased ROS generation and NOX4 gene expression, and inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI) and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC is added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with siRNA decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3-K) signaling pathway inhibitor LY294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3-K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-1 that acts in an autocrine fashion to induce IGFBP-3. IGFBP-3 through PI3-K/Akt increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.