Display omitted •TSPO is over-expressed and correlates oxidative stress and the mPTP opening in cardiomyocytes after A/R injury.•Downregulation of TSPO by RNAi specifically reduces oxidative stress.•Downregulation of TSPO stabilizes the mitochondrial membrane potential and inhibits mPTP opening.•Downregulation of TSPO expression prevents apoptosis in A/R-exposed cardiomyocytes. Translocator protein (TSPO) is highly expressed in the cardiovascular system, exerting crucial effects on both myocardial damage and protection. However, the role and mechanism of TSPO in myocardial ischemia/reperfusion (I/R) injury remains elusive. In the current study, we subjected H9c2 cardiomyocytes to anoxia/reoxygenation (A/R) and knocked down TSPO expression by RNA interference to investigate the possible mechanism of TSPO on I/R injury. TSPO expression in cardiomyocytes was up-regulated when exposed to A/R, but normal in anoxic preconditioned (APC) cardiomyocytes. Moreover, A/R also led to an increase in reactive oxygen species (ROS), oxidative stress aggravation, mitochondrial membrane potential collapse, mitochondrial permeability transition pore (mPTP) opening, and cell apoptosis. However, these events were completely compensated by downregulating TSPO expression. TSPO-downregulated cardiomyocytes produced lesser lactate dehydrogenase (LDH) and creatine phosphokinase (CPK), and showed lesser cytosol malondialdehyde (MDA) accumulation than normal cells after A/R injury. On the other hand, the TSPO- downregulated cells showed higher activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), mitochondrial function stabilization, resulting in less cell apoptosis and damage in case of A/R condition. In conclusion, TSPO expression is up-regulated under A/R injury, whereas repression of TSPO improves the endurance of cardiomyocytes against A/R injury by reducing oxidative stress, mitochondrial damage and cell apoptosis.