Sepsis leads to the damage of multiple organs, and thereby adversely affects the cardiovascular system. At present, no effective method has been found to treat myocardial injury caused by sepsis. While Puerarin was reported to attenuate LPS-induced mitochondrial injury in HUVECs, the effects of Puerarin in sepsis-induced myocardial injury remain unclear. In this study, H9C2 cells were stimulated with LPS, CCK-8 assays were performed to assess cell viability, and flow cytometry and TUNEL staining were used to assess cell apoptosis. Levels of ATP, ADP, AMP and enzyme activity were investigated using commercial kits. ROS levels in H9C2 cells were detected by flow cytometry. Autophagosomes in the mitochondria of H9C2 cells were observed by transmission electron microscope (TEM), and protein expression was assessed by western blotting. We found that Puerarin significantly reversed LPS-induced decreases in H9C2 cell viability by inhibiting apoptosis. The ROS levels in H9C2 cells were significantly upregulated by LPS, but that effect was markedly reduced by Puerarin. In addition, Puerarin attenuated LPS-induced mitochondrial injury in H9C2 cells by regulating dynamin-related protein 1 (Drp1) and mitofusin 1 (Mfn1). LPS decreased enzyme activity and reduced the levels of ADP, ALP, and AMP in mitochondria; however, those effects were reversed by Puerarin. Puerarin and Torin1 reversed LPS-induced inhibition of autophagy in the mitochondria of H9C2 cells via mediation of p62, LC3B, Pink1, and Parkin. Puerarin inhibited LPS-induced H9C2 cell injury by inducing mitochondrial autophagy, which acts as a mechanism for preventing myocardial injury caused by sepsis.Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.