Mitochondria-derived methylmalonic acid (MMA) aggravates ischemia-reperfusion (I/R) injury by activating reactive oxygen species (ROS)-dependent ferroptosis. This study investigated the role of MMA in ferroptosis activation in cardiomyocytes following I/R. MMA levels were elevated in patients with acute myocardial injury after reperfusion and in AC16 cells under hypoxia/reoxygenation (H/R) conditions. MMA treatment increased oxidative stress and ferroptosis-related myocardial injury. In mice with I/R injury, MMA treatment exacerbated myocardial oxidative stress and ferroptosis, increasing infarct size and cardiac dysfunction. Mechanistically, MMA promoted NOX2/4 expression, increasing ROS production and aggravating myocardial injury. Increased ROS further activated ferroptosis by inhibiting SLC7A11 and GPX4 expression. MMA also decreased the nuclear distribution of NRF2 by enhancing its interaction with KEAP1, impairing the activation of GPX4/SLC7A11 and activating ferroptosis. These findings suggest that MMA activates oxidative stress and ROS generation, inducing ferroptosis and exacerbating cardiomyocyte injury in an I/R model. These results may provide new insights for the clinical treatment of I/R injury.Mitochondria-derived methylmalonic acid (MMA) aggravates ischemia-reperfusion (I/R) injury by activating reactive oxygen species (ROS)-dependent ferroptosis. This study investigated the role of MMA in ferroptosis activation in cardiomyocytes following I/R. MMA levels were elevated in patients with acute myocardial injury after reperfusion and in AC16 cells under hypoxia/reoxygenation (H/R) conditions. MMA treatment increased oxidative stress and ferroptosis-related myocardial injury. In mice with I/R injury, MMA treatment exacerbated myocardial oxidative stress and ferroptosis, increasing infarct size and cardiac dysfunction. Mechanistically, MMA promoted NOX2/4 expression, increasing ROS production and aggravating myocardial injury. Increased ROS further activated ferroptosis by inhibiting SLC7A11 and GPX4 expression. MMA also decreased the nuclear distribution of NRF2 by enhancing its interaction with KEAP1, impairing the activation of GPX4/SLC7A11 and activating ferroptosis. These findings suggest that MMA activates oxidative stress and ROS generation, inducing ferroptosis and exacerbating cardiomyocyte injury in an I/R model. These results may provide new insights for the clinical treatment of I/R injury.