Abstract: Objective: To investigate the mechanisms of GSH interaction with Bcl-2 following one-time exercise precondition(EP) in reducing cardiotoxicity induced by DOX treatment of cancer. Methods: Forty 19-week-old female SD rats were randomly divided into control group(C), doxorubicin group(DOX), exercise preconditioning group(EP), and exercise preconditioning + doxorubicin group(EP+DOX), 10 rats in each group. The Dox group rats were injected with doxorubicin, EP group rats were conducted about of high-intensity intermittent exercise, and the EP+DOX group rats were injected with doxorubicin 24 h after EP. Cardiac ultrasound was used to detect cardiac function in rats; chemical fluorescence immunoassay was used to detect the expression level of myocardial injury markers; histological staining and transmission electron microscopy were used to observe and analyze the histopathological changes of myocardium; Western blot was used to detect myocardial apoptosis and pro-survival gene-related protein expression; Metabolomics analysis was performed to explore the changes of myocardial metabolites and metabolic pathways;immunoprecipitation was used to detect the interaction between Bcl-2 and OGC(GSH transporter protein); immunofluorescence double-labeling was used to analyze the co-localization of GSH and Bcl-2. Results: In the DOX group, the myocardial mitochondria were swollen, the outer membrane was ruptured, the volume increased, and the mitochondrial cristae were disorganized or even broken; in the mitochondrial matrix, the linear dense cristae were visible, and the cloudy flocculent structures were appeared.Compared with group C, the cardiac function of rats in the DOX group was significantly decreased; the serum c Tn I, LDH, and MDA contents of myocardial injury indexes were significantly higher; the symptoms of myocardial cell injury, fibrosis, and inflammation were obvious; the expression levels of anti-apoptotic protein Bcl-2 and cardiomyocytes were significantly lower, and the expression of apoptotic proteins such as Bad, Bax and Caspase3 were significantly higher; YAP phosphorylation level was significantly increased; GSH and NAD+ metabolism level were significantly lower; Bcl-2 and OGC interaction was significantly lower; Bcl-2 and GSH co-localization level was significantly lower. Compared with the DOX group, the EP+DOX group showed improvements in all of the above indicators. Conclusions: The classical EP can attenuate DOX treatment-induced apoptosis and oxidative stress injury in cardiac myocytes by promoting GSH/Bcl-2 interaction, and it also can inhibit inflammation and fibrosis,and improve cardiac function.