The inhibition of tumor angiogenesis is one of the main challenges in cancer therapy. With the aim of developing monoclonal antibodies able to inhibit angiogenesis, we immunized mice with proliferating human umbilical vein endothelial cells. We generated a library of monoclonal antibodies able to recognize antigens expressed on endothelial cells and screened the antibodies for their ability to inhibit endothelial cell proliferation, migration, and sprouting in vitro. Here, we show that the antibody, designated as 4E1, is able to neutralize the formation of new vessels both in vitro and in vivo without affecting endothelial cell survival. By mass spectrometry we identified CD93 as the antigen bound by 4E1 and mapped the recognized epitope. CD93 is a transmembrane protein heavily glycosylated preferentially expressed in the vascular endothelium. CD93 silencing by lentiviral-mediated small hairpin RNA expression impairs human endothelial cell proliferation, migration, and sprouting. Altogether these findings reveal 4E1 as a novel antiangiogenic antibody and identify CD93 as a new target suitable for antiangiogenic therapy.