Hydrogen sulfide (H₂S) reduces myocardial ischemia-reperfusion (I-R) injury by preserving mitochondrial function. This study demonstrates that H₂S delivery at reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of I-R. The protective effect is associated with reduced myocardial inflammation and preserved mitochondrial structure and function. Overexpression of cystathionine γ-lyase (CGL) in cardiac-specific transgenic mice also limits I-R injury, indicating that H₂S may be beneficial in cytoprotection during myocardial infarction. H₂S administration or modulation of endogenous production could have clinical relevance in ischemic disorders. H₂S is an endogenously produced gaseous second messenger that modulates physiological processes, similar to nitric oxide. It is produced by two enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL), which are heme-containing enzymes dependent on the cofactor pyridoxal 5'-phosphate (PLP). H₂S has been shown to regulate N-methyl-D-aspartate receptors and may be central in long-term potentiation of neuronal circuitry. It is also produced in the vasculature by CGL, where it mediates smooth muscle relaxation and vasodilation independent of the GC/cGMP pathway. In this study, H₂S donor therapy at reperfusion significantly reduced infarct size and preserved LV function in mice. H₂S treatment reduced myocardial inflammation, preserved mitochondrial function, and decreased cardiomyocyte apoptosis. H₂S also reduced neutrophil infiltration, MPO activity, and inflammatory cytokines such as IL-1β. Intravital microscopy showed that H₂S inhibited thrombin-induced leukocyte-endothelial cell interactions. In vitro studies showed that H₂S dose-dependently inhibited cardiac mitochondrial respiration and preserved mitochondrial function. H₂S-treated mitochondria showed improved recovery of respiration rate after hypoxia and increased complex I and II efficiency. Electron microscopy revealed preserved mitochondrial structure and function in H₂S-treated mice. Cardiac-specific overexpression of CGL increased myocardial H₂S production and reduced infarct size in mice. These findings suggest that H₂S may be a promising therapeutic agent for acute myocardial infarction. The study highlights the potential of H₂S as a cardioprotective agent through its effects on mitochondrial function and inflammation.Hydrogen sulfide (H₂S) reduces myocardial ischemia-reperfusion (I-R) injury by preserving mitochondrial function. This study demonstrates that H₂S delivery at reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of I-R. The protective effect is associated with reduced myocardial inflammation and preserved mitochondrial structure and function. Overexpression of cystathionine γ-lyase (CGL) in cardiac-specific transgenic mice also limits I-R injury, indicating that H₂S may be beneficial in cytoprotection during myocardial infarction. H₂S administration or modulation of endogenous production could have clinical relevance in ischemic disorders. H₂S is an endogenously produced gaseous second messenger that modulates physiological processes, similar to nitric oxide. It is produced by two enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL), which are heme-containing enzymes dependent on the cofactor pyridoxal 5'-phosphate (PLP). H₂S has been shown to regulate N-methyl-D-aspartate receptors and may be central in long-term potentiation of neuronal circuitry. It is also produced in the vasculature by CGL, where it mediates smooth muscle relaxation and vasodilation independent of the GC/cGMP pathway. In this study, H₂S donor therapy at reperfusion significantly reduced infarct size and preserved LV function in mice. H₂S treatment reduced myocardial inflammation, preserved mitochondrial function, and decreased cardiomyocyte apoptosis. H₂S also reduced neutrophil infiltration, MPO activity, and inflammatory cytokines such as IL-1β. Intravital microscopy showed that H₂S inhibited thrombin-induced leukocyte-endothelial cell interactions. In vitro studies showed that H₂S dose-dependently inhibited cardiac mitochondrial respiration and preserved mitochondrial function. H₂S-treated mitochondria showed improved recovery of respiration rate after hypoxia and increased complex I and II efficiency. Electron microscopy revealed preserved mitochondrial structure and function in H₂S-treated mice. Cardiac-specific overexpression of CGL increased myocardial H₂S production and reduced infarct size in mice. These findings suggest that H₂S may be a promising therapeutic agent for acute myocardial infarction. The study highlights the potential of H₂S as a cardioprotective agent through its effects on mitochondrial function and inflammation.