Although both alcohol and fructose are particularly steatogenic, their long-term effect in the development of a metabolic syndrome has not been studied in vivo. Consumption of fructose generally leads to obesity, whereas ethanol can induce liver damage in the absence of overweight. Here, Sprague-Dawley rats were fed ad libitum for 28 days on five diets: chow (control), liquid Lieber-DeCarli (LDC) diet, LDC +30%J of ethanol (L-Et) or fructose (L-Fr), and LDC combined with 30%J ethanol and 30%J fructose (L-EF). Body weight (BW) and liver weight (LW) were measured. Blood and liver samples were harvested and subjected to biochemical tests, histopathological examinations, and RT-PCR. Alcohol-containing diets substantially reduced the food intake and BW (≤3rd week), whereas fructose-fed animals had higher LW than controls (P<0.05). Additionally, leukocytes, plasma AST and leptin levels were the highest in the fructose-administered rats. Compared to the chow and LDC diets, the L-EF diet significantly elevated blood glucose, insulin, and total-cholesterol levels (also vs. the L-Et group). The albumin and Quick-test levels were the lowest, whereas ALT activity was the highest in the L-EF group. Moreover, the L-EF diet aggravated plasma triglyceride and reduced HDL-cholesterol levels more than 2.7-fold compared to the sum of the effects of the L-Et and L-Fr diets. The decreased hepatic insulin clearance in the L-EF group vs. control and LDC groups was reflected by a significantly decreased C-peptide:insulin ratio. All diets except the control caused hepatosteatosis, as evidenced by Nile red and H&E staining. Hepatic transcription of insulin receptor substrate-1/2 was mainly suppressed by the L-Fr and L-EF diets. The L-EF diet did not enhance the mitochondrial β-oxidation of fatty acids (Cpt1α and Ppar-α expressions) compared to the L-Et or L-Fr diet. Together, our data provide evidence for the coaction of ethanol and fructose with a high-fat-diet on dyslipidemia and insulin resistance-accompanied liver damage.