Background: Ginsenoside Rb1, a bioactive component isolated from the Panax ginseng, acts as a remedyto prevent myocardial injury. However, it is obscure whether the cardioprotective functions of Rb1 arerelated to the regulation of endogenous metabolites, and its potential molecular mechanism still needsfurther clarification, especially from a comprehensive metabolomics profiling perspective. Methods: The mice model of acute myocardial ischemia (AMI) and oxygen glucose deprivation (OGD)-induced cardiomyocytes injury were applied to explore the protective effect and mechanism of Rb1. Meanwhile, the comprehensive metabolomics profiling was conducted by high-performance liquidchromatography and quadrupole time-of-flight mass spectrometry (HPLC-Q/TOF-MS) and a tandemliquid chromatography and mass spectrometry (LC-MS). Results: Rb1 treatment profoundly reduced the infarct size and attenuated myocardial injury. Themetabolic network map of 65 differential endogenous metabolites was constructed and provided a newinspiration for the treatment of AMI by Rb1, which was mainly associated with mitophagy. In vivo andin vitro experiments, Rb1 was found to improve mitochondrial morphology, mitochondrial function andpromote mitophagy. Interestingly, the mitophagy inhibitor partly attenuated the cardioprotective effectof Rb1. Additionally, Rb1 markedly facilitated the phosphorylation of AMP-activated protein kinase a(AMPKa), and AMPK inhibition partially weakened the role of Rb1 in promoting mitophagy. Conclusions: Ginsenoside Rb1 protects acute myocardial ischemia injury through promoting mitophagyvia AMPKa phosphorylation, which might lay the foundation for the further application of Rb1 in cardiovascular diseases.