Mequindox (MEQ), a member of Quinoxaline 1, 4–di–N–oxides (QdNOs), has been applied in Scophthalmus maximus farming industry. Due to the severe toxic effects of QdNOs, the residue of MEQ and its metabolites in Scophthalmus maximus are food safety risk factors. In this work, metabolism of MEQ in Scophthalmus maximus in vivo was investigated. Eight metabolites were identified to be formed via hydrogenation reduction, desoxygenation, deacetylation, methyl mono–hydroxylation, and their combination reactions. Different from land farm animal with desoxygenation as major metabolic reaction, carbonyl reduction is the major metabolic reaction in Scophthalmus maximus. In addition, M8 (3–methylol–BDMEQ) was first discovered in aquatic organisms with methyl mono–hydroxylation as a novel metabolic reaction, and M7 (Deacetyl–4–DMEQ) was the first metabolite to be detected in an organism undergoing both N→O deoxygenation and deacetylation reactions. This work revealed metabolic reactions of MEQ in Scophthalmus maximus and provided the process to study this metabolism in other marine animals.