Tumor angiogenesis is a critical process during cancer progression that modulates tumor growth and metastasis. Here, we identified an anti-angiogenic microRNA, miR-320, which is decreased in oral squamous cell carcinoma (OSCC) cell lines and tumor tissues from OSCC patients, down-regulated in blood vessels and inversely correlated with vascularity in OSCC tissues. Neuropilin 1 (NRP1), an important regulator of angiogenesis, was found to be a target of miR-320. The 3'-untranslated region of NRP1 mRNA contains multiple miR-320 binding sites, and its expression was regulated by miR-320. By administering either miR-320 precursor or antagonist, we found that miR-320 suppressed the migration, adhesion and tube formation of vascular endothelial cells. Knockdown of NRP1 abolished antagomiR-320-induced cell migration. Additionally, miR-320 expression was regulated by hypoxia in growth factor-deficient conditions by the hypoxia-inducible factor 1-alpha. Furthermore, lentivirus carrying the miR-320 precursor suppressed the tumorigenicity of OSCC cells and tumor angiogenesis in vivo. Taken together, these data show that miR-320 regulates the function of vascular endothelial cells by targeting NRP1 and has the potential to be developed as an anti-angiogenic or anti-cancer drug.