The prevalence of diabetic cardiomyopathy is increasing at an alarming rate, it is therefore urgent to identify potential therapeutic targets. Impairment of glycolytic metabolism is suggested to contribute to diabetic cardiomyopathy. Our previous studies showed that the expression of Sirtuin 3 (Sirt3) was significantly reduced which was associated with microvascular rarefaction and cardiac dysfunction in diabetic db/db mice and obesity mice. Our recent study indicated a critical role of endothelial Sirt3 derived glycolysis in the diastolic dysfunction. So far, little is known about Sirt3 in diabetes impairment of cardiomyocyte glycolytic metabolism. Using cultured cardiomyocytes exposed to high glucose (HG, 30 mM) and diabetic db/db mice, we explored the roles of Sirt 3 on cardiomyocyte metabolism and cardiac function by transfection with adenovirus‐Sirt3 (Ad‐Sirt3). We found that exposure of cardiomyocytes to HG resulted in a gradual decrease in Sirt3 expression. This was accompanied by upregulation of p53 acetylation and expression. Transfection with Ad‐Sirt3 significantly reduced hyperglycemia‐induced p53 acetylation and expression. Using XFe24 Extracellular Flux analyzer, we examined the glycolytic metabolism of cardiomyocyte under HG conditions. The basal glycolysis, glycolytic capacity and glycolytic reserve were significantly reduced in the cardiomyocyte exposed to HG. The basal oxygen consumption rate and levels of ROS formation were significantly increased in the cardiomyocyte exposed to HG, indicating hyperglycemia shifted glycolytic metabolism to oxidative metabolism. Overexpression of Sirt3 increased the expression of HIF‐1α and glycolytic enzyme PFKFB3, rescued impaired glycolysis and reduced mitochondrial oxygen consumption. Hyperglycemia‐induced ROS formation, caspase‐3 expression and apoptosis was also significantly suppressed by Sirt3. Knockdown of p53 in cardiomyocytes by using siRNA significantly attenuated hyperglycemia‐induced impairment of the expression of HIF‐1α, PFKFB3 and VEGF. Treatment with p53 siRNA reprogramed metabolism by increasing glycolysis and decreasing oxygen consumption. In vivo, an intravenous jugular vein injection of Ad‐Sirt3 in diabetic db/db mice resulted in a significant increase in Sirt3 expression with a reduction of p53 acetylation in the heart. Ad‐Sirt3 treatment upregulated the expression of HIF‐1α, PFKFB3 and VEGF. Immuno‐histologic studies found that Ad‐Sirt3 treatment increased angiogenesis, and reduced ROS formation and apoptosis in the diabetic heart. Importantly, Ad‐Sirt3 treatment significantly improved cardiac function (LVSD, LVSV, EF%, FS%) measured by the echocardiography. This study suggests that Sirt3 may be a novel target for abnormal energy metabolism in diabetic cardiomyopathy.Support or Funding InformationThis study was supported by grants from NIH grant 2R01HL102042‐05 and University of Mississippi Medical Center Intramural Research Support Program to J.X. Chen.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.