© 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Endoglin is a transforming growth factor β (TGF-β) co-receptor essential for angiogenesis and tumor vascularization. Endoglin modulates the crucial balance between pro- and anti-angiogenic signaling by activin receptor-like kinase (ALK) 1, 5, and TGF-β type II (TβRII) receptors. Despite its established role in physiology and disease, the mechanism of endoglin down-regulation remains unknown. Herewereport that the conserved juxtamembrane cytoplasmic tyrosine motif (612YIY614) is a critical determinant of angiogenesis. Src directly phosphorylates this motif to induce endoglin internalization and degradation via the lysosome. We identified epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as Src-activators that induce endoglin turnover following 612YIY614phosphorylation. Interestingly, Src phosphorylation of endoglin-612YIY614was also an important process for receptor down-regulation by TRACON105 (TRC105), an endoglin-targeting antibody currently in clinical trials. The regulation of 612YIY614phosphorylation was critical for angiogenesis, as both the phosphomimetic and unphosphorylatable mutants impaired endothelial functions including proliferation, migration, and capillary tube formation. Collectively, these findings establish Src and pro-angiogenic mitogens as critical mediators of endoglin stability and function.