Aim Previous studies have indicated that H₂S can attenuate myocardial fibrosis. However, it is unclear whether mitochondria-targeted H₂S can attenuate myocardial fibrosis after myocardial infarction and whether its mechanism is associated with the regulation of mitochondrial fusion and fission. To investigate this relationship, this study was conducted. Methods Isoproterenol (ISO, 50 mg/(kg·d) was injected intraperitoneally to induce myocardial infarction in SD rats. Electrocardiograms were performed on each group of rats, and the rats were treated with AP39 (36 μg/(kg·d), intraperitoneal) for 4 weeks. Masson's staining was used to assess the extent of myocardial fibrosis. Western blot was used to measure the expression of relevant proteins. In vitro experiments were performed to induce hypoxic injury in H9c2 cardiomyocytes with CoCl₂ (800 μmol/L), H9c2 cells were treated with AP39 (100 nmol/L), and the endogenous hydrogen sulfide synthase cystathionine-γ-lyase(CSE) was inhibited using DL-propargylglycine (PAG, 2 mmol/L), and fluorescence probe was used to measure the level of reactive oxygen species(ROS) in myocardial cells. Results Myocardial fibrosis was evident in infarcted rat hearts, with a significant accumulation of collagen fibers. Additionally, the expression of CSE and mitofusin 2 (MFN2) proteins was downregulated, while dynamin-related protein 1(DRP1) protein expression was increased. Intervention with AP39 significantly improved the above changes, and the addition of CSE inhibitor PAG reversed the effects of AP39. In in vitro experiments, when H9c2 myocardial cells were subjected to hypoxic injury induced by CoCl₂, intracellular ROS levels increased, MFN2 expression was downregulated, and DRP1 expression was upregulated. AP39 upregulated MFN2 protein expression, inhibited DRP1 protein expression, and reduced ROS levels in myocardial cells. The addition of PAG reversed these changes. Conclusion The mitochondria-targeted H₂S donor, AP39, can improve myocardial fibrosis in rats with myocardial infarction and promote mitochondrial fusion and inhibit excessive mitochondrial division.