Abstract Excessive exercise leads to myocardial injury or even sudden exercise death. For the vast sports population, appropriate physiological state is a necessary condition for exercise. The present study aims to investigate the cardioprotective effects and potent mechanism of astragalus polysaccharide (APS) treatment against the exercise-induced myocardial injury via in vitro cell-based assay and in vivo model rat. Efficacies of APS incubation on the inflammatory response and oxidative stress induced by LPS were both explored in H9c2 cells by using CCK-8 and western blotting method, respectively. Normal SD rats were randomly divided into saline-treated overexercise rat group, and APS-treated overexercise rat groups with three doses. Then long-term swimming training load cycle (8 week) were performed on these rats. Finally, the changes on body weight, myocardial morphological and injury indicators, as well as the inflammation-related proteins in overexercise-induced model rats were all assessed. Three concentrations of APS all significantly increased cell viability, and decreased the apoptosis of cardiomyocytes in LPS-treated H9c2 cells. Moreover, chronic treatment of APS at all three doses also could obviously decreased myocardial injury-related indicators. Furthermore, the histopathologic examination exhibited that the APS successfully attenuated the changes of myocardial tissues, reduced the lipid accumulation and the protein levels of IL-1β, TNF-α and NF-κB. Furthermore, the APS could activate the AMPK signaling pathway, enhance the autophagy and suppress the production of ROS. On conclusions, APS exerted the protective efficacies on overexercise-induced myocardial injury by activating the AMPK signaling pathway to increase autophagy and suppress the inflammation response, oxidative stress, apoptosis of myocardial cells.