As the frequency of global heat waves keeps rising, exertional heat stroke (EHS) is becoming an increasingly prevalent health concern, which causes myocardial injury. However, the mechanisms underlying myocardial injury following EHS are still unclear. In this study, we established an EHS mouse model in which mice were exercised by running on a treadmill in a high-humidity and high-temperature environment and investigated the time points of myocardial injury within 24 h after EHS. We found that mice had cardiac dysfunction and myocardial injury after EHS and that the damage was the most serious at 6 h of recovery. Next, changes in cardiac ferroptosis and lipid peroxidation levels after EHS were evaluated, and ferroptosis was found to be the main form of myocardial cell death, and inhibition of ferroptosis by liproxstatin-1 ameliorated EHS-induced myocardial injury. In addition, we found that arachidonic acid 15-lipoxygenase-1 (Alox15) is a critical molecule of ferroptosis in cardiomyocytes through targeted metabolomics experiments. Based on in vivo and in vitro studies, inhibiting Alox15 conspicuously ameliorates EHS-induced cardiac dysfunction and myocardial injury. Mechanistically, EHS-induced excessive activation of transcription factor p53 upregulated Alox15 expression via inducing SAT1 (spermidine/spermine N1-acetyltransferase 1) expression and reduced the expression of Gpx4 (glutathione peroxidase 4) to initiate ferroptosis. This study reveals the key role of ferroptosis in EHS-induced myocardial injury and confirms that Alox15-mediated ferroptosis of cardiomyocytes is the core pathological mechanism. This finding provides a new molecular target and theoretical basis for the prevention and treatment strategies of EHS-related myocardial injury.