Aim To investigate whether modified Zhishi Xiebai Guizhi decoction activates MitoKATP channel during myocardial ischemia-reperfusion to produce mitochondrial protection, thereby inhibiting mitochondrial apoptosis pathway and alleviating myocardial ischemia-reperfusion injury. Methods Twenty-four Sprague-Dawley rats were randomly divided into 3 groups:sham operation group (n=6), ischemia reperfusion model group (IR group, n=9), modified Zhishi Xiebai Guizhi decoction group (M group, n=9). After 14 days of administration, coronary artery anterior descending was performed. The model was established, and the ST segment of myocardial electrocardiogram, myocardial enzyme (CK-MB, LDH) and mitochondrial ultrastructural changes were observed. The myocardial infarct size of IR group and M group was compared by Evans blue/TTC staining. After rhodamine 123 staining, the mitochondrial membrane potential was detected by flow cytometry. The expressions of connexin43 (Cx43), protein kinase C-ε (PKC-ε) and inward-rectifier potassium channel 6.2 (Kir6.2) subunits in mitochondria of each group were detected by Western blot. Methylthymol blue microplate assay was used to detect calcium content in mitochondria of rats in each group. Results Modified Zhishi Xiebai Guizhi decoction can effectively alleviate ST segment and myocardial enzyme changes caused by myocardial ischemia-reperfusion, reduce myocardial infarct size, reduce mitochondrial ultrastructure and membrane potential changes, enhance the expression of Cx43, PKC-ε and Kir6.2 in mitochondrial and mitigating mitochondrial calcium overload. Conclusion Modified Zhishi Xiebai Guizhi decoction can activate MitoKATP channel to activate mitochondrial protective effect by up-regulating the expression of Cx43 and PKC-ε in mitochondria, thereby reducing the release of cytochrome C from mitochondria, inhibiting the cascade response of caspase-related proteins to reduce myocardial cells apoptosis rate, and to reduce myocardial ischemia-reperfusion injury.