Neovascularization, the formation of new blood vessels, is fundamental to cardiac repair and regeneration in ischemic heart disease. After myocardial infarction (MI), vascular stem cells (VSC) are mobilized from bone marrow and recruited to the ischemic site, de novo generating new blood vessels to promote cardiac recovery. Our previous studies have revealed that phosphorylation of profilin-1 (Pfn-1) induces endothelial cell migration and sprouting angiogenesis. Here, we show that Pfn-1 phosphorylation regulates VSC homing to ischemic site and cardiac repair after MI through protease expression. Vascular lineage-specific knock-in of phosphorylation-dead Pfn-1(Y129F) mutant in mice show that Pfn-1 phosphorylation is critical for ischemia-induced neovascularization and cardiac function recovery after MI. Deficiency in Pfn-1 phosphorylation inhibits VSC homing to the ischemic hindlimb, suggesting a critical role of Pfn-1 phosphorylation in VSC recruitment. Mechanistic studies show that Pfn-1 phosphorylation is required for vascular endothelial growth factor (VEGF)-A-induced expression of metalloproteinase (MMP) −2 and −9 and cell migration in VSC. Therefore, these studies identify a critical role of Pfn-1 phosphorylation in VSC homing and neovascularization after MI, and suggest that Pfn-1 phosphorylation may represent as a therapeutic target for treating ischemic heart disease.