To investigate the effect of hydrogen-rich water on myocardial tissue metabolism in a myocardial ischemia-reperfusion injury (MIRI) rat model. Twelve rats were randomly divided into a hydrogen-rich water group and a control group of size 6 each. After the heart was removed, it was fixed in the Langendorff device, and the heart was perfused with 37 degrees C perfusion solution pre-balanced with oxygen. The control group was perfused with Kreb's-Ringers (K-R) solution, and the hydrogen-rich water group was perfused with K-R solution + hydrogen-rich water. Liquid Chromatograph Mass Spectrometer (LC-MS) analysis platform was used for metabolomics research. Principle component analysis (PCA), partial least squares discriminant analysis (PLS-DA), orthogonal partial least squares discriminant analysis (OPLS-DA), Variable importance in projection (VIP) value of OPLS-DA model (threshold value >= 1) were employed with independent sample T Test (p < 0.05) to find differentially expressed metabolites, and screen for differential metabolic pathways. VIP (OPLS-DA) analysis was performed with T test, and the metabolites of the control group and the hydrogen-rich water group were significantly different, and the glycerophospholipid metabolism was screened. Seven myocardial ischemia-reperfusion injury (MIRI)-related signaling pathways were identified, including glycerophospholipid metabolism, glycosylphosphatidylinositol (GPI) anchored biosynthesis, and purine metabolism, as well as 10 biomarkers such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Hydrogen-rich water regulates the metabolic imbalance that could change MIRI myocardial tissue metabolism, and alleviate ischemia-reperfusion injury in isolated hearts of rats through multiple signaling pathways.