Objective— Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. Approach and Results— A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1 ^−/− when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1 ^−/− lung endothelial cells (LECs) and a more tip-cell–like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1 ^−/− LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1 ^−/− LECs when compared with wild-type LECs. Conclusions— NoxO1 stimulates α-secretase activity probably through reactive oxygen species–mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.