Uncontrolled proliferation of endothelial cells is essential to the pathogenesis of pulmonary arterial hypertension (PAH). Both proliferation and cytoskeleton reorganization are associated with upregulation of the intermediate filament protein Nestin. Recently, accumulation of Nestin-expressing cells was found in pulmonary vascular lesions of PAH patients. The goal of this study is to determine if Nestin expression contributes to endothelial proliferation in pulmonary arterial hypertension, using both lung tissues and endothelial cells. Here we found that endothelial cells from complex and plexiform lesions of PAH patients expressed Nestin. These Nestin+ cells further stained positive for the angiogenic factors CXC chemokine ligand 12 and Wnt1. Likewise, in the chronic hypoxia/SU5416 animal model of pulmonary hypertension, Nestin+ endothelial cells were found in occlusive pulmonary vascular lesions. In vitro, both growing rat and human lung endothelial cells expressed Nestin protein. When Nestin was overexpressed in endothelial cells (both rat and human), Nestin overexpression promoted proliferation and expression of CXC chemokine ligand 12. Nestin overexpression further increased angiogenic tube formation in vitro. Conclusions: We found increased Nestin expression from endothelial cells of occlusive lung vascular lesions in severe pulmonary hypertension. Elevated Nestin expression likely contributes to unchecked pulmonary vascular proliferation and angiogenesis, possibly via induction of CXC chemokine ligand 12. Additional studies are required to determine whether targeting Nestin would be beneficial to treat PAH.