Extra domain-B containing fibronectin (EDB⁺ FN), a recently proposed marker of angiogenesis, has been shown to be expressed in a number of human cancers and in ocular neovascularization in patients with proliferative diabetic retinopathy. To gain molecular understanding of the functional significance of EDB⁺ FN, we have investigated possible regulatory mechanisms of induction and its role in endothelial cell proliferation and angiogenesis. Human vascular endothelial cells were cultured in high levels of glucose, and fibrogenic growth factors, transforming growth factor-β1 (TGF-β1) and endothelin-1 (ET-1). Our results show that high glucose levels, TGF-β1, and ET-1 upregulated EDB⁺ FN expression. Treatment of cells exposed to high glucose with TGF-β1 neutralizing antibody and ET receptor antagonist prevented high glucose-induced EDB⁺ FN expression. In order to elucidate the functional significance of EDB⁺ FN upregulation, cells were subjected to in vitro proliferation and angiogenesis assays following EDB peptide treatment and specific EDB⁺ FN gene silencing. Our results show that exposure of cells to EDB peptide increased vascular endothelial growth factor (VEGF) expression, endothelial proliferation, and tube formation. Furthermore, specific EDB⁺ FN gene silencing prevented both basal and high glucose-induced VEGF expression and reduced the proliferative capacity of endothelial cells. In conclusion, these results indicate that EDB⁺ FN is involved in endothelial cell proliferation and vascular morphogenesis, findings which may provide novel avenues for the development of anti-angiogenic therapies.