GRS is a drug combination of three active components including ginsenoside Rb1, ruscogenin and schisandrin. It derived from the well-known TCM formula ShengMai preparations, a widely used traditional Chinese medicine for the treatment of cardiovascular diseases in clinic. The present study explores the cardioprotective effects of GRS on myocardial ischemia/reperfusion (MI/R) injury compared with ShengMai preparations and investigates the underlying mechanisms. GRS treatment significantly attenuated MI/R injury and exhibited similar efficacy as Shengmai preparations, as evidenced by decreased myocardium infarct size, ameliorated histological features, the decrease of LDH production and improved cardiac function, and also produced a significant decrease of apoptotic index. Mechanistically, GRS alleviated myocardial apoptosis by inhibiting the mitochondrial mediated apoptosis pathway as reflected by inhibition of caspase-3 activity, normalization of Bcl-2/Bax levels and improved mitochondrial function. Moreover, GRS prevented cardiomyocytes mitochondrial fission and upregulated AMPKα phosphorylation. Interestingly, AMPK activation prevented hypoxia and reoxygenation induced mitochondrial fission in cardiomyocytes and GRS actions were significantly attenuated by knockdown of AMPKα. Collectively, these data show that GRS is effective in mitigating MI/R injury by suppressing mitochondrial mediated apoptosis and modulating AMPK activation-mediated mitochondrial fission, thereby providing a rationale for future clinical applications and potential therapeutic strategy for MI/R injury.