Diabetic cardiomyopathy is a major diabetic complication featured by the impaired angiogenesis and cardiac dysfunction. Apelin (APLN) has been shown to prompt angiogenesis. Our recent study implicates a critical role of sirtuin 3 (SIRT-3) in the regulation of angiopoietins expression and angiogenesis. In the present study, we investigated the roles of SIRT-3 in APLN-induced angiogenesis in a diabetic cardiomyopathy model underwent ischemia. Male wild type (WT) mice and SIRT-3 knock-out (SIRT-3 KO) mice were induced into diabetic cardiomyopathy model by STZ injection and followed by left anterior descending artery ligation to induce myocardial infarction (MI). The experimental mice then underwent intra-myocardium injection with adenovirus Apelin (Ad -APLN) or β-gal adenovirus (Ad-β-gal) immediately after the ligation. Myocardial angiogenesis and cardiac function was measured 2 weeks after surgery. Our data revealed that the ejection fraction (EF) and fraction shorting (FS) were significantly improved in STZ mice received Ad-APLN treatment than in STZ mice received Ad-β-gal (EF: 64.1% ±3.2 vs 52.8%±5.6, P<0.001; FS: 34.3±2.2% vs, 26.6±3.5%, P<0.001). Ad-APLN treatment in STZ-SIRT3KO mice had little effects on EF and FS (EF: 47.9±5.2% vs, 49.7±8.2%, P=0.634; FS: 24.7±5.1% vs, 23.5±3.1%, P=0.728). The expressions of angiopoietins-1 and angiopoietins-2 were significantly increased in Ad-APLN treated STZ mice compared to Ad-β-gal treated STZ mice. These were accompanied by significant increases in myocardial arterioles and capillary densities. Ad-APLN mediated angiopoietins expressions and neovascularization were completely abolished in STZ-SIRT3 KO mice. In vitro, Ad-APLN significantly enhanced the proliferation and tube formation of endothelial cells derived from WT mice, but failed to do that in cells from SIRT 3 KO mice. Our data suggest that SIRT-3 is essential for the beneficial effect of Apelin on angiogenesis and heart function in diabetic cardiomyopathy.