Myocardial infarction (MI) is a type of cardiovascular disease that influences millions of human beings worldwide and has a great rate of mortality and morbidity. Spironolactone has been used as a critical drug for the treatment of cardiac failure and it ameliorates cardiac dysfunction post-MI. Despite these findings, whether there is a relationship between the therapeutic effects of spironolactone and the gut microorganism after MI has not been determined. In our research, we used male C57BL/6 J mice to explore whether the gut microbiota mediates the beneficial function of spironolactone after myocardial infarction. We demonstrated that deletion of the gut microbiota eliminated the beneficial function of spironolactone in MI mice, displaying exacerbated cardiac dysfunction, cardiac infarct size. In addition, the gut microbiota was altered by spironolactone after sham or MI operation in mice. We also used male C57BL/6 J mice to investigate the function of a probiotic in the myocardial infarction. In summary, our findings reveal a precious role of the gut flora in the therapeutic function of spironolactone on MI.