Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-α (Dgk-α) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-α is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-α: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-α forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-α, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-α dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-α by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-α in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-α generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.