Revascularization is an effective therapy for rescuing myocardial tissue after ischemic events. However, the process of reperfusion can lead to more severe cardiomyocyte damage, called myocardial ischemia-reperfusion (I/R) injury (MIRI). We have previously shown that vitexin (VT) (a flavonoid compound derived from natural products) protects against MIRI; however, the exact mechanisms underpinning this effect require further elucidation. This study is aimed at elucidating the protective mechanism of VT in inhibiting ischemic myocardial mitochondrial dysfunction and reducing cardiomyocyte apoptosis by regulating Epac1-Rap1 signaling. Isolated rat hearts were subjected to MIRI in a Langendorff perfusion system, and H9c2 cells were subjected to hypoxia/reoxygenation (H/R) in vitro. Our analyses show that during I/R, Epac1 expression was upregulated, left ventricular dysfunction deteriorated, mitochondrial dynamics were disrupted, and both myocardial cells and tissues exhibited apoptosis. Furthermore, administration of 8-CPT (an Epac agonist) exacerbated cardiomyocyte injury and mitochondrial dysfunction. Interestingly, suppressing the function of Epac1 through VT or ESI-09 (an Epac inhibitor) treatment during I/R reduced the myocardial infarct size, cardiomyocyte apoptosis, and reactive oxygen species production; alleviated mitochondrial dysfunction by increasing mitochondrial membrane potential; elevated MFN2 expression; and inhibited Drp1 expression. To our knowledge, our results reveal, for the first time, the mechanisms underlying the protective effect of VT in the myocardium of rats with MIRI. Moreover, we provide a new target and theoretical basis for VT in the treatment of ischemic heart disease.