BACKGROUND: Acute myocardial infarction (AMI) is the primary cause of cardiac mortality worldwide. However, myocardial ischemia-reperfusion injury (MIRI) following reperfusion therapy is common in AMI, causing myocardial damage and affecting the patient's prognosis. Presently, there are no effective treatments available for MIRI. METHODS: We performed a comprehensive bioinformatics analysis using three GEO datasets on differentially expressed genes, including gene ontology (GO), pathway enrichment analyses, and protein-protein interaction (PPI) network analysis. Cytoscape and LASSO methods were employed to identify novel regulator genes for ischemia-reperfusion (I/R). Notably, gene S100A9 was identified as a potential regulator of I/R. Additionally, clinical sample datasets were analyzed to prove the expression and mechanism of S100A9 and its down genes in I/R. The correlation of S100A9 with cardiac events was also examined to enhance the reliability of our results. RESULTS: We identified 135 differential genes between the peripheral blood of 47 controls and 92 I/R patients. S100A9 was distinguished as a novel regulator gene of I/R with diagnostic potential. RT-qPCR test demonstrated significant upregulation of S100A9 in I/R. We also verified that S100A9 expression strongly correlates with left ventricular ejection fraction (LVEF) and MIRI. CONCLUSION: This study confirms that S100A9 is a key regulator of I/R progression and may participate in ischemia-reperfusion injury by upregulating RAGE /NFKB-NLRP3 activation. Elevated S100A9 levels may serve as a marker for identifying high-risk MIRI patients, especially those with coronary artery no-reflow (CNR), who might benefit from targeted therapeutic interventions. Furthermore, Peripheral blood S100A9 in AMI represents a new therapeutic target for preventing MIRI.