Gentianella acuta (Michx.) Hulten is widely used for the treatment of arrhythmia and coronary heart disease in Ewenki Folk Medicinal Plants and Mongolian Medicine, popularly known as “Wenxincao” in China. To investigate the potential protective role of the xanthones from G. acuta against myocardial I/R injury in isolated rat heart and its possible related mechanism. The protective role of xanthones on myocardial I/R injury was studied on Langendorff apparatus. The hemodynamic parameters including the left ventricular developed pressure (LVDP), the maximum rate of up/down left intraventricular pressure (±dp/dt_max), coronary flow (CF) and heart rate (HR) were recorded during the perfusion. The results demonstrated that the xanthones from G. acuta treatment significantly improved myocardial function (LVDP, ±dp/dt_max and CF), increased the levels of superoxide dismutase (SOD) and catalase (CAT), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), ATP and the ratio of glutathione and glutathione disulfide (GSH/GSSG), whereas suppressed the levels of Lactate dehydrogenase (LDH), creatine kinase (CK) and malondialdehyde (MDA). Furthermore, the xanthones upregulate the level of Bcl-2 protein and downregulate the level of Bax protein. These results indicated that xanthones from G. acuta exhibited cardioprotective effects on myocardial I/R injury through its activities of anti-oxidative effect and anti-apoptosis effect.