Mitochondria are the primary source of cellular energy, but they also damage cells by producing reactive oxygen species (ROS). Peroxiredoxin III (Prx III), a mitochondrial-specific H₂O₂ scavenging enzyme, protects against mitochondrial dysfunction by removing excessive mitochondrial ROS. Doxorubicin (DOX) is an anthracycline anticancer drug that is known to have cardiotoxicity. Since cardiomyocytes have much higher ATP synthesis and oxygen consumption than other cells, mitochondrial injury is a significant cause of heart failure. Hypoxia/reoxygenation (H/R) is also the main cause of heart failure due to ROS generation. In this study, we investigated how Prx Ⅲ protects against role of with both DOX- and H/R-induced cardiotoxicity by keeping autophagy flux and lysosome activity high. We measured autophagy flux, lysosome activity and acidity, and lysosome-autophagosome colocalization. After DOX or H/R treatment, autophagy flux was disrupted due to a lack of lysosomal activity in Prx Ⅲ-deficient cells. After reconstitution of Prx III by adenovirus, autophagy flux was recovered with normal lysosome activity. Thus, we demonstrated that Prx III effectively protects cardiomyocytes through proper autophagy.