Hyperglycemia is associated with a reduced number of endothelial progenitor cells (EPCs) that impairs vascular function. Circulating EPCs play important roles in postnatal neovasculogenesis and the prevention of ischemic injury. Frequent consumption of fish oil (FO) that is abundant with eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) is reportedly associated with an alleviation of diabetic complications and a lowered incidence of cardiovascular disease. The aim of this study was to examine whether N-3 polyunsaturated fatty acids such as EPA and DHA would reverse the high glucose-mediated dysfunction of EPCs in vitro and thereby prevent the ischemic injury that occurs under the hyperglycemic conditions in Type 2 diabetes (T2D) db−/− mice. The results demonstrate that EPA and DHA alleviate high glucose-mediated impairment of tubular formation in EPCs through a rescue of neovasculogenic capability. The molecular mechanisms underlying the effects of EPA and DHA include the activation of the extracellular signal-regulated kinase 1/2, Akt/endothelial nitric oxide synthase (eNOS) and AMP-activated kinase (AMPK) signaling cascades as well as the phosphorylation of the downstream FOXO3a protein in EPCs. Moreover, EPA and DHA up-regulate the expression of c-kit, erythroid 2-related factor and heme oxygenase-1 proteins. Daily consumption of FO at dosages of 4% and 6% (wt/wt) significantly increased the level of bone marrow-derived and circulating EPCs, induced a recovery of blood flow and prevented ischemic injuries in a T2D db−/− mouse model. The effects of FO consumption were exerted the activation of Akt/eNOS and AMPK signaling cascades without any effect on the plasma VEGF level in vivo.