AIMS: Luteolin is a falconoid compound that has an antioxidant effect, but its contribution to ROS-activated MAPK pathways in ischemia/reperfusion injury is seldom reported. Here, we have confirmed that it exhibits an antioxidant effect in myocardial ischemia/reperfusion injury (MIRI) by inhibiting ROS-activated MAPK pathways. MAIN METHODS: We exposed rat hearts into the left anterior descending coronary artery (LAD) ligation for 30min followed by 1h of reperfusion. Observations were carried out using electrocardiography; detection of hemodynamic parameters; and testing levels of lactate dehydrogenase (LDH), creatine kinase (CK), total superoxide dismutase (T-SOD), and malondialdehyde (MDA). Mitogen-activated protein kinase (MAPK) pathway was measured by western blot and transmission electron microscopy was applied to observe the myocardial ultrastructure. Rat H9c2 cell in 95% N2 and 5% CO2 stimulated the MIRI. Oxidation system mRNA levels were measured by real-time PCR; mitochondrial membrane potential and apoptosis were measured by confocal microscopy and flow cytometry; western blot analysis was used to assay caspase-3, -8, and -9 and MAPK pathway protein expression; the MAPK pathway was inhibited using SB203580 (p38 MAPK inhibitor) and SP600125 (c-Jun NH2-terminal kinase inhibitor) before H9c2 cells were exposed to hypoxia/reoxygenation injury to show the modulation of the changes in ROS generation, cell viability and apoptosis. KEY FINDINGS: In vivo, luteolin can ameliorate the impaired mitochondrial morphology, regulating the MAPK pathway to protect MIRI. In vitro, luteolin can affect the oxidation system, mitochondrial membrane potential and MAPK pathway to anti-apoptosis. SIGNIFICANCE: These results reveal a ROS-MAPK mediated mechanism and mitochondrial pathway through which luteolin can protect myocardial ischemia/reperfusion injury.