Background Myocardial ischemic/reperfusion (I/R) injury is a key prognostic factor after the myocardial infarction. However, at the time of reperfusion, the myocardial tissue has undergone for the necrosis and initiated the induction of oxidative stress and inflammation. The current study was to scrutinize the cardioprotective effect of gossypin against ISO-induced I/R injury in myocardial tissue and explore the possible underlying mechanism. Methods Sprague Dawley (SD) was used in the current protocol and ISO was used for induction the I/R in rat. The rats were divided into different groups and received the oral administration of gossypin treatment before the reperfusion. The body weight, heart weight and heart body weight ratio were estimated. The antioxidant, cardiac injury parameters, inflammatory cytokines, inflammatory mediators, gut microbiota and lipid parameters were estimated. At the end, heart tissue histopathological study was carried out. Results ISO-induced I/R rats received the gossypin treatment significantly (P < 0.001) enhanced the body weight and decreased the heart weight, along with suppressed the infarct size. Gossypin treatment significantly (P < 0.001) reduced the level of heart parameters, such as creatinine kinase-MB (CK-MB), lactate dehydrogenase (LDH), creatine kinase (CK), cardiac troponin I (CTn-I) and cardiac troponin T (CTn-T) in the serum. Gossypin treatment significantly (P < 0.001) altered the cardiac function, hepatic, antioxidant, inflammatory cytokines and inflammatory mediators. Gossypin significantly (P < 0.001) suppressed the MMP-2 and MMP-9 in ISO-induced I/R rats. Gossypin treatment considerably alleviated the gut dysbiosis through altered Firmicutes to Bacteroidetes (F/B) ratio and also maintained the relative abundance of Butyricicoccus, Clostridium IV, Akkermansia, Roseburia and Clostridium XIVs. Conclusion Based on result, we can conclude that gossypin is an alternative drug for the treatment of ISO-induced I/R in rats via alteration of oxidative stress, inflammatory reaction and gut microbiota.