Abstract Body (Do not enter title and authors here): Introduction: Myocardial ischemia-reperfusion (IR) injury is the most common type of heart disease. IR-disrupted mitochondrial homeostasis affects heart energy metabolism and function, but the mechanism remains poorly understood. Lactylation is a novel type of protein PTM involved in the metabolic regulation of gene expression and function. In this study, we uncover an essential role of Sirt5 in maintaining cardiac mitochondrial homeostasis by regulating lysine lactylation modification of adenine nucleotide translocase 2 (ANT2). Hypothesis:Sirt5 protects mitochondrial and cardiac functions. Methods: Sirt5 floxed mice were injected with AAV9 encoding cTnT-driven Cre recombinase to knockout (KO) Sirt5 in cardiomyocytes (CMs). CM-specific Sirt5 KO mice, Sirt5 floxed mice, AAV9-Ctrl mice, AAV9-Sirt5 mice, were subjected to 45 minutes of I/R surgery, and the wild-type littermates was used as a control to determine the cardiac function of the group. We used 4D label-free proteomics technology to identify differentially lactylated proteins in mouse ventricular myocardial tissues following IR injury and Sirt5-interacting proteins in HL-1 cells. Results: In HL-1 cells, Sirt5 knockdown exacerbated mitochondrial fragmentation and ROS levels, while overexpression protected against these effects (n=5, P<0.05 for all). In vivo, Sirt5 overexpression in C57BL/6 mice improved heart function (ejection fraction: P<0.01, n=5; fractional shortening: P<0.05, n=5), reduced infarct size (P<0.01, n=5), and decreased inflammatory cell infiltration compared to sham group. Conversely, Sirt5 knockout worsened these parameters (n=5, P<0.05 for all). These findings highlight Sirt5's role in maintaining mitochondrial and cardiac function post-IR injury. Mechanistically, Sirt5 interacts with adenine nucleotide translocase 2 (ANT2), inhibiting its lysine lactylation at residue K163, thereby promoting its association with voltage-dependent anion-selective channel 1 (VDAC1) on the outer mitochondrial membrane. This interaction facilitates metabolite exchange between the cytosol and mitochondrial matrix, maintaining mitochondrial homeostasis. A lactylation-resistant ANT2 mutant (ANT2-K163R) efficiently complexes with VDAC1 and rescues mitochondrial dysfunction and cardiac injury caused by IR or Sirt5 deficiency. Conclusions: Sirt5 preserves mitochondrial homeostasis to improve cardiac function in ischemia-reperfusion injury by inhibiting ANT2 lysine lactylation modification