This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H₂O₂) in glutathione peroxidase-1 (gpx-1^−/−) and catalase (catalase^−/−) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1^−/− displayed significantly higher hepatic H₂O₂ levels than catalase^−/− compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1^−/− mice. Alcohol increased H₂O₂ production in catalase^−/− and wild-type, but not gpx-1^−/−, mice. Hepcidin expression was inhibited in alcohol-fed catalase^−/− and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1^−/− mice. Gpx-1^−/− mice also displayed higher level of basal liver CHOP protein expression than catalase^−/− mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1^−/− mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1^−/− mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H₂O₂ inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H₂O_2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH. Full article