Ischemia - reperfusion is a common cause of myocardial injury, and there are currently no clinically approved drugs for its treatment. Oxidative stress caused by mitochondrial ROS is the main cause of cell damage in ischemia - reperfusion (I/R). Therefore, exploring methods to reduce mitochondrial ROS during I/R is essential. Here, we investigated the effects of a low concentration of the uncoupler FCCP on hypoxia/reoxygenation (H/R) injury in myocardial cells. An in vitro myocardial I/R model was constructed by using sodium sulfite (Na ₂ SO ₃ ) to induce hypoxia and then reoxygenation. We found that after hypoxia, treatment with 5 nM FCCP induced the expression of UCP1, which uncouples mitochondria, thereby decreasing ATP production, ROS levels, and mitophagy, ultimately reducing myocardial injury. In vivo experiments further revealed that 1 mg/kg body weight FCCP has a protective effect against myocardial I/R injury. These data indicated that mildly uncoupling mitochondria via a low concentration of FCCP attenuated I/R-triggered cardiac injury.