ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non-phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H2O2 on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti-angiogenesis. Our results showed that H2O2 dose-dependently increased the generation of O2 − (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H2O2 at low concentrations (10 µM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H2O2 at 4 nmol/cm2 strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm2). Also, H2O2 (200∼500 µM) could stimulate apoptosis in HUVECs. All the effects of H2O2 on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H2O2 to produce O2 − and then to regulate angiogenesis. In summary, our results suggest that H2O2 as well as O2 − mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.