The present study aimed to investigate the protective effects and molecular mechanisms of scoparone on ischemia-reperfusion (I/R) injury in primary cultured cardiac myocytes and rats. An in vivo rat model of I/R injury and an in vitro primary cultured cardiac myocyte model of oxygen-glucose deprivation/reoxygenation were used to investigate the protective effects of scoparone. Cell viability, lactate dehydrogenase (LDH) release, superoxide dismutase (SOD), creatine kinase (CK) and malondialdehyde (MDA) levels, and reactive oxygen species (ROS) production were subsequently measured. In addition, cell apoptosis was assessed by terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining, and myocardial infarct area (IA) was determined by triphenyl tetrazolium chloride staining. Furthermore, the protein expression levels of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cytochrome c (Cyt C) and caspase-3 were assessed by western blotting. The results demonstrated that treatment with scoparone markedly increased cell viability, SOD levels and Bcl-2 protein expression, and decreased LDH release, MDA production, CK levels, ROS concentration, cell apoptotic rate, myocardial IA, and Bax, caspase-3 and Cyt C protein expression. These findings indicated that scoparone may have a protective effect against I/R injury, thus suggesting that scoparone may be a considered a potential drug for the treatment of I/R injury via the inhibition of oxidative stress and cell apoptosis.