Objective To investigate the impact of exogenous gaseous signaling molecule sulfur dioxide (SO₂) on myocardial fibrosis in rat models of type 2 diabetes mellitus (T2DM), and to observe its effects on expressions of pro-apoptotic proteins and proteins associated with the MST1/LATS1 pathway.Methods Forty male SD rats were randomly divided into the control group, diabetes mellitus group, diabetes mellitus + SO₂ group, and SO₂ group. For rats in the diabetes mellitus group and the diabetes mellitus + SO₂ group, diabetes mellitus was induced by intraperitoneal injection of streptozotocin (STZ) and confirmed by the blood glucose concentration ≥ 16.7 mmol/L. After successful model establishment, rats were fed with a high-sugar and high-fat diet. The rats in the control group and the SO₂ group were injected with an equal volume of citric acid-sodium citrate solution. Subsequently, the rats in the diabetes mellitus + SO₂ group and the SO₂ group were intraperitoneally injected with exogenous SO₂ for 4 weeks. Myocardial tissues from the rats were subjected to various analyses, including Masson staining to assess collagen fiber formation, transmission electron microscopy to observe myocardial ultrastructure, TUNEL staining to evaluate apoptosis, and Western blotting to measure protein expression levels of collagen I, collagen III, MST1, LATS1, cleaved Caspase-3, and cleaved Caspase-9.Results In comparison to the control group, the diabetes mellitus group exhibited larger positive areas on Masson staining (P < 0.05). The positive areas on Masson staining in the diabetes mellitus + SO₂ group were smaller than those in the diabetes mellitus group (P < 0.05). There was no difference in the positive areas on Masson staining between the control group and the SO₂ group (P > 0.05). The relative protein expression levels of collagen I and collagen III in the diabetes mellitus group were higher than those in the control group (P < 0.05), while they were lower in the diabetes mellitus + SO₂ group than those in the diabetes mellitus group (P < 0.05). There was no difference in relative protein expression levels of collagen I and collagen III between the control group and the SO₂ group (P > 0.05). The apoptosis rate of cardiomyocytes in the diabetes mellitus group was higher than that in the control group (P < 0.05), while that in the diabetes mellitus + SO₂ group was lower compared with the diabetes mellitus group (P < 0.05). There was no difference in the apoptosis rate of cardiomyocytes between the control group and the SO₂ group (P > 0.05). The relative protein expression levels of cleaved Caspase-3, cleaved Caspase-9, MST1 and LATS1 in the myocardial tissues of the diabetes mellitus + SO₂ group were lower than those of the diabetes mellitus group (P < 0.05). There was no difference in the relative protein expression levels of cleaved Caspase-3, cleaved Caspase-9, MST1 and LATS1 between the control group and the SO₂ group (P > 0.05).Conclusions SO₂ regulates the expressions of pro-apoptotic proteins and proteins associated with the MST1/LATS1 pathway in myocardial tissues of rat models of T2DM, and exhibits a correlation with the amelioration of myocardial fibrosis.