In adults, angiogenic abnormalities are involved in not only tumor growth but several human inherited diseases as well. It is unclear, however, concerning how the normal vascular structure is maintained and how angiogenesis is initiated in normal adults. Using the Cre‐LoxP‐mediated conditional gene deletion, we show in the present study that in adult mice disruption of the transcription factor recombination signal‐binding protein Jκ (RBP‐J) in endothelial cells strikingly induced spontaneous angiogenesis in multiple tissues, including retina and cornea, as well as in internal organs, such as liver and lung. In a choroidal neovascularization model, which mimics the angiogenic process in tumor growth and age‐related macular degeneration, RBP‐J deficiency induced a more intensive angiogenic response to injury. This could be transmitted by bone marrow, indicating that RBP‐J could modulate bone marrow‐derived endothelial progenitor cells in adult angiogenesis. In addition, in the absence of RBP‐J, proliferation of endothelial cells increased significantly, leading to accumulative vessel outgrowth. These findings suggest that in adults RBP‐J‐mediated Notch signaling may play an essential role in the maintenance of vascular homeostasis by repressing endothelial cell proliferation.—Dou, G.‐R., Wang, Y.‐C., Hu, X.‐B., Hou, L.‐H., Wang, C.‐M., Xu, J.‐F., Wang, Y.‐S., Liang, Y.‐M., Yao, L.‐B., Yang, A.‐G., Han, H. RBP‐J, the transcription factor downstream of Notch receptors, is essential for the maintenance of vascular homeostasis in adult mice. FASEB J. 22, 1606–1617 (2008)