<p>Myocardial ischemia–reperfusion injury (MIRI) is an injury mechanism of myocardial infarction, related to ferroptosis and glycolysis. Lactate produced by glycolysis promotes protein lactylation. This study aimed to investigate the correlation between glycolysis, ferroptosis, and GPX4 lactylation in MIRI. Hypoxia/reoxygenation (H/R) increased glucose uptake, lactate production, ECAR, OCR, LDH release, lipid ROS, Fe²⁺<b>,</b> GSH, MDA contents, and cell apoptosis, and decreased GSH level in the H9C2 cells, suggesting H/R promoted glycolysis and ferroptosis. 2-DG treatment relieved the H/R-induced injury, while lactate treatment aggravated it. Besides, 2-DG suppressed lactylation of GPX4 at K218 and K228 sites and increased its protein stability. GPX4 overexpression relieved the injury caused by H/R, and alleviated cardiac injury, decreased cardiomyocyte ferroptosis in heart tissues of MIRI rats. In conclusion, GPX4 lactylation facilitated H/R-induced cardiomyocyte injury and aggravated MIRI in rats. Our findings provided new insight into targeting glycolysis and GPX4 lactylation as therapeutic strategies of MIRI.</p> Graphical abstract <p></p>