Alcohol reduces and iron increases liver hepcidin synthesis. This study investigates the interaction of alcohol and iron in the regulation of hepcidin messenger RNA (mRNA) expression in animal models. Mice were administered 10% ethanol for 7 days after an iron‐overloaded diet. Rats were administered regular or ethanol‐Lieber De Carli diets for 7 weeks with or without carbonyl iron. Hfe^−/− mice were used as a model for genetic iron overload. Hepcidin mRNA expression was determined by real‐time polymerase chain reaction (PCR) and northern blotting. Iron elevated and alcohol decreased liver hepcidin expression in mice and rats. Interestingly, despite iron overload, alcohol was capable of suppressing the up‐regulation of hepcidin mRNA expression in both models. Liver iron and ferritin protein expression was elevated in alcohol‐treated rats, but was not elevated further in rats treated with both iron and alcohol. Duodenal ferroportin protein expression was increased both in alcohol‐treated mice and in mice treated with alcohol and iron. Hfe^−/− mice treated with ethanol for 7 days exhibited a further decrease in hepcidin mRNA expression. The iron‐induced increase in DNA‐binding activity of the transcription factor CCAAT/enhancer binding protein alpha (C/EBP alpha) was also suppressed by alcohol. Conclusion: Alcohol abolishes the iron‐induced up‐regulation of both liver hepcidin transcription and the DNA‐binding activity of C/EBP alpha. Of note, hepcidin protects the body from the harmful effects of iron overload. Our findings therefore suggest that alcohol negates the protective effect of hepcidin, which may have implications for the liver injury observed in alcoholic liver disease and genetic hemochromatosis in combination with alcohol. (HEPATOLOGY 2007.)