Myocardial ischemia-reperfusion injury (MIRI) is a type of severe injury to the ischemic myocardium that can occur following recovery of blood flow, and for which, there is no effective treatment. Panaxatriol saponin (PTS), a major active component of P. notoginseng, has been used clinically to treat ischemia-related encephalopathy due to its antioxidant activity, but its effect on ischemic cardiomyopathy and underlying mechanism of action is still unclear. This study was performed to investigate the protective effect of PTS against MIRI and explore the potential underlying mechanisms. Hydrogen peroxide (H2O2) was used to stimulate cardiomyocytes, to mimic MIRI in vitro. Cell viability was tested using the CCK-8 method. The antioxidant activity of PTS in the H9c2 rat cardiomyocyte cell line was examined using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA). The levels of superoxide dismutase-1 (SOD1), SOD2, and heme oxygenase (HO-1) were determined by Western blotting and/or immunofluorescence. The antiapoptotic effect of PTS was determined. In addition, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential () changes were assessed. Changes in Keap1/Nrf2 activation were evaluated by Western blotting analysis, molecular docking, and immunoprecipitation. An in vivo MIRI model was established in rats, and the myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Myocardial enzyme activities were determined by ELISA or biochemical analyses. Furthermore, changes in Nrf2 activation were evaluated, and the regulatory effect of PTS on cardiomyocyte apoptosis was examined using the Nrf2 blocker, ML385. The results showed that PTS ameliorated the cardiomyocyte injury induced by H2O2, characterized by increased cell viability, decreased reactive oxygen species (ROS) production, and promotion of SOD1, SOD2, and HO1 expression. PTS inhibited cardiomyocyte apoptosis in vivo and in vitro. PTS also reduced mPTP opening and stabilized in H9c2 cells. Molecular docking and immunoprecipitation study revealed that PTS can disrupt Keap1/Nrf2 interaction by directly blocking the binding site of Nrf2 in the Keap1 protein. In vivo, PTS decreased the area of myocardial infarction and attenuated pathological damage in ischemia-reperfusion (I/R) rats. In addition, the activities of myocardial injury markers were decreased by PTS. Finally, PTS regulated nuclear translocation of Nrf2, and ML385 blocked the therapeutic effect of PTS in vivo and in vitro. These results suggested that PTS has therapeutic potential for MIRI by targeting Keap1/Nrf2 activity.