CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146EC-KO) mice using the Tg(Tek–cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146EC-KO mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells (ECs) of CD146EC-KO mice. Mechanistic studies further confirmed that VEGF-induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/NF-kB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146EC-KO mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.