Ischemia/reperfusion (I/R) injury is a pathological process caused by reperfusion. The prevention of I/R injury is of great importance as it would enhance the efficacy of myocardial infarction treatment in patients. Isovaleroylbinankadsurin A (ISBA) has been demonstrated to possess multiple bioactivities for treating diseases. However, its protective effect on myocardial I/R injury remains unknown. In this study, the cardiomyocytes hypoxia/reoxygenation (H/R) in vitro model and coronary artery ligation in vivo model were used to examine the protective effect of ISBA. Apoptosis was determined by flow cytometry and Caspase 3 activity. Protein level was determined by Western blot. The mitochondrial viability was examined with mitochondrial viability stain assay. Mitochondrial membrane potential was detected by JC-1 staining and reactive oxygen species (ROS) was stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). The binding interactions between ISBA and receptors was simulated by molecular docking. Results showed that ISBA effectively protected cardiomyocytes from I/R injury in in vitro and in vivo models. It remarkably blocked the apoptosis induced by H/R injury through the mitochondrial dependent pathway. Activation of the reperfusion injury salvage kinase (RISK) pathway was demonstrated to be essential for ISBA to exert its protective effect on cardiomyocytes. Moreover, molecular docking indicated that ISBA directly bound to and activated glucocorticoid receptor (GR), and GR inhibitor RU486 partially counteracted the protective effect of ISBA on cardiomyocytes. Most attractively, by activating GR dependent RISK pathway, ISBA significantly elevated the cellular anti-oxidative capacity and hence alleviated oxidative damage induced by I/R injury. In conclusion, our study proved that ISBA protected the heart from myocardial I/R injury through activating GR dependent RISK pathway and consequently inhibiting the ROS generation. It provides a valuable reference for ISBA to be developed as a candidate drug for cardiovascular diseases.Copyright © 2020. Published by Elsevier Ltd.