Background: The differentially expressed proteins (DEPs) involved in the effect of hydrogen-rich water on myocardial ischemia reperfusion injury (MIRI) and their biological processes and signaling pathway were analyzed. Methods: 20 Wistar rats were randomly and equally divided into a control and a hydrogen-rich group. Hearts were removed and fixed in a Langendorff device. The control group was perfused with K-R solution, and the hydrogen-rich water group was perfused with K-R solution + hydrogen-rich water. Protein was extracted from the ventricular tissues, and GSR-CAA-67 was used to identify the DEPs between two groups. DEPs were analyzed through bioinformatic methods. Results: Compared with the control group, in the treatment group, the expression of 25 proteins was obviously decreased (P<0.05). For the DEPs, 359 biological processes, including the regulation of signaling pathways, immune reaction and formation of cardiovascular endothelial cells, were selected by GO enrichment analysis. Five signaling pathways were selected by KEGG pathway enrichment analysis. Conclusions: 25 proteins that are involved in hydrogen-water reducing MIRI were selected by high-throughput GSR-CAA-67. The biological processes and metabolic pathways involved in the DEPs were summarized, providing theoretical evidence for the clinical application of hydrogen-rich water.