Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show that VEGF-A-inducible phosphorylation of Pfn-1 at Tyr 129 is critical for endothelial cell migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induces Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Conditional endothelial knock-in of phosphorylation-deficient Pfn1(Y129F) in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischaemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signalling events to initiate endothelial cell migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy.