BACKGROUND: Alcohol abuse and adherence to atherogenic diet (AD; a low-carbohydrate-high-protein diet) have been positively associated with cardiovascular disease. In addition, it has been demonstrated clinically that dietary intake is increased on days when alcohol is consumed. Here, the additive effects of ethanol (EtOH) and AD on atherosclerosis, a major underlying cause of cardiovascular disease, were investigated in apolipoprotein E/low-density lipoprotein receptor double-knockout (KO) mice. The mechanisms, especially aortic oxidative stress damage, were highlighted. METHODS: Twelve-week-old male KO mice on AD with or without EtOH treatment were bred for 4 months. Age-matched male C57BL/6J mice on a standard chow diet without EtOH treatment served as controls. Analyses were conducted using ultrasound biomicroscopy, histopathological and fluorescence immunohistochemical examinations, Western blots, and polymerase chain reaction. RESULTS: KO mice on AD with EtOH treatment showed increases in aortic maximum intima media thickness, hypoechoic plaque formation, and mean Oil-Red-O content. These results were associated with enhanced ratio of aortic 8-hydroxy-2'-deoxyguanosine (8-OHdG)-immunopositive area to the metallothionein (MT) immunopositive area and suppression of AD-induced up-regulated aortic Mt1, Mt2, and upstream stimulatory factor 1 mRNA expressions. Moreover, 8-OHdG was expressed in the nuclei of CD31- and alpha smooth muscle actin-immunopositive cells, and the up-regulated mRNA expressions of aortic nitric oxide synthase 3 and platelet-derived growth factors were only observed in the KO mice on AD with EtOH treatment. CONCLUSIONS: Alcohol abuse and adherence to AD may promote the shift of aortic oxidative stress and antioxidative stress balance toward oxidative stress predominance and reduced antioxidative stress, which may be partly due to the decrease in MT at the cell biological level and down-regulation of Mt at the gene level, which in turn could play a role in the up-regulation of endothelial dysfunction-related and vascular smooth muscle cell proliferation-related gene expression and the progression of atherosclerosis in mice with hyperlipidemia.