Hydrogen sulfide (H2S) is involved in diverse physiological functions, such as anti-hypertension, anti-proliferation, regulating ATP synthesis, and reactive oxygen species production. Sirtuin 3 (SIRT3) is a NAD + -dependent deacetylase that regulates mitochondrial energy metabolism. The role of H2S in energy metabolism in diabetic cardiomyopathy (DCM) may be related to regulate SIRT3 expression; however, this role remains to be elucidated. We hypothesized that exogenous H2S could switch cardiac energy metabolic substrate preference by lysine acetylation through promoting the expression of SIRT3 in cardiac tissue of db/db mice. Db/db mice, neonatal rat cardiomyocytes, and H9c2 cell line with the treatment of high glucose, oleate, and palmitate were used as animal and cellular models of type 2 diabetes. Using LC-MS/MS, we identified 76 proteins that increased acetylation, including 8 enzymes related to fatty acid β-oxidation and 7 enzymes of the tricarboxylic acid (TCA) cycle in the db/db mice hearts compared to those with the treatment of NaHS. Exogenous H2S restored the expression of NAMPT and the ratio of NAD+/NADH enhanced the expression and activity of SIRT3. As a result of activation of SIRT3, the acetylation level and activity of fatty acid β-oxidation enzyme LCAD and the acetylation of glucose oxidation enzymes PDH, IDH2, and CS were reduced which resulted in activation of PDH, IDH2, and CS. Our finding suggested that H2S induced a switch in cardiac energy substrate utilization from fatty acid β-oxidation to glucose oxidation in DCM through regulating SIRT3 pathway.