Mitochondrial aldehyde dehydrogenase (ALDH2) protects against cardiac injury via reducing production of 4-hydroxynonenal (4-HNE) and ROS. This study was designed to examine the impact of ALDH2 on doxorubicin (DOX)-induced cardiomyopathy and mechanisms involved with a focus on autophagy. 4-HNE and autophagic markers were detected by Western blotting in ventricular tissues from normal donors and patients with idiopathic dilated cardiomyopathy. Cardiac function, 4-HNE and levels of autophagic markers were detected in WT, ALDH2 knockout or ALDH2 transfected mice treated with or without DOX. Autophagy regulatory signaling including PI-3K, AMPK and Akt was examined in DOX-treated cardiomyocytes incubated with or without ALDH2 activator Alda-1. DOX-induced myocardial dysfunction, upregulation of 4-HNE and autophagic proteins were further aggravated in ALDH2 knockout mice while they were ameliorated in ALDH2 transfected mice. DOX downregulated Class I and upregulated Class III PI3-kinase, the effect of which was augmented by ALDH2 deletion. Accumulation of 4-HNE and autophagic protein markers in DOX-induced cardiomyocytes was significantly reduced by Alda-1. DOX depressed phosphorylated Akt but not AMPK, the effect was augmented by ALDH2 knockout. The autophagy inhibitor 3-MA attenuated, whereas autophagy inducer rapamycin mimicked DOX-induced cardiomyocyte contractile defects. In addition, rapamycin effectively mitigated Alda-1-offered protective action against DOX-induced cardiomyocyte dysfunction. Our data further revealed downregulated ALDH2 and upregulated autophagy levels in the hearts from patients with dilated cardiomyopathy. Taken together, our findings suggest that inhibition of 4-HNE and autophagy may be a plausible mechanism underscoring ALDH2-offered protection against DOX-induced cardiac defect. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy".