Human endothelial progenitor cells (hEPCs) derived from bone marrow play a crucial in the prevention of ischemic injury in the course of postnatal neovasculogenesis. Frequent fish oil (FO) consumption is reportedly associated with a significantly lower incidence of cardiovascular disease (CVD). However, the molecular mechanisms of eicosapentaenoic acid (EPA) / docosahexaenoic acid (DHA) are not well elucidated, and the beneficial effect of FO consumption on neovasculogenesis has not been demonstrated yet. In the current study, we investigated the effects of EPA/DHA and FO consumption on neovasculogenesis both in vitro and in vivo. The results demonstrate that EPA /DHA dose‐dependently enhance the neovasculogenesis and cell migration of hEPCsin vitro. The mechanisms of action included upregulation of the c‐kit protein as well as the phosphorylation of the ERK1/2, Akt and endothelial nitric oxide synthase (eNOS) signaling molecules in hEPCs. Furthermore, EPA significantly suppressed the expression of microRNA (miR) 221 in vitro. In experimental animal models, FO consumption significantly induced the formation of new blood vessels (neovasculogenesis) and prevented ischemia. Taken together, it is suggested that FO consumption enhances neovasculogenesis mainly through the effects of EPA in hEPCs, thereby exerting a preventive effect against ischemic injury.