Angiogenesis is essential many physiological and pathological events, for instance in wound repair and tumor progression. The origin of newly-formed blood vessel endothelial cells in adults is not known. Here we identified and isolated c-kit+ vascular endothelial stem cells (VESCs) by the phenotype lin-CD31+CD105+Sca1+CD117+, a small subpopulation residing on the vessel wall. A single transplanted VESC is able to generate functional blood vessels in connection with the host circulation and maintain a long-term self-renewal capacity in vivo. Further, we showed that a genetic deficit in endothelial c-kit expression remarkably decreases total colony-forming VESCs, indicating a functional role of c-kit in VESCs. A deficit in c-kit expression also resulted in impaired angiogenesis and growth retardation in tumor. Thus, VESCs would provide a novel cellular target to inhibit pathological angiogenesis in cancer therapies. Isolated VESCs could also be utilized in therapies to restore tissue vascularization in many diseases.