Reactive oxygen species (ROS)‑induced mitochondrial dysfunction plays an important role in cardiomyocyte apoptosis during myocardial ischemia/reperfusion (I/R) injury. Picroside Ⅱ, isolated from Picrorhiza scrophulariiflora Pennell (Scrophulariaceae), has been reported to protect cardiomyocytes from hypoxia/reoxygenation (H/R)‑induced apoptosis, but the exact mechanism is not fully clear. The aim of the present study was to explore the protective effects of picroside Ⅱ on H/R‑induced cardiomyocyte apoptosis and the underlying mechanism. In the H9c2 rat cardiomyocyte cell line, picroside Ⅱ (100 µg/ml) was added for 48 h prior to H/R. The results showed that picroside Ⅱ markedly inhibited H/R‑induced cardiomyocyte apoptosis. In addition, picroside Ⅱ was also able to decrease the opening degree of mitochondrial permeability transition pore (mPTP), increase the mitochondrial membrane potential, inhibit cytochrome c release from mitochondria to cytosol and downregulate caspase‑3 expression and activity concomitantly with the decreased ROS production. These results suggested that picroside Ⅱ inhibited H/R‑induced cardiomyocyte apoptosis by ameliorating mitochondrial function through a mechanism involving a decrease in ROS production.