Abstract Mequindox, 3‐methyl‐2‐quinoxalinacetyl‐1,4‐dioxide, is a quinoxaline‐ N , N ‐dioxide used in veterinary medicine as a antibacterial in China. To gain an understanding of the interspecies differences in the metabolism of mequindox, comparative metabolite profiles were qualitatively and quantitatively carried out for the first time in rat, chicken and pig liver microsomes by high‐performance liquid chromatography combined with hybrid ion trap/time‐of‐flight mass spectrometry. A total of 14 metabolites were characterized based on their accurate MS 2 spectra and known structure of mequindox. The in vitro metabolic pathways of mequindox in three species were proposed as N→O group reduction, carbonyl reduction, N→O group reduction followed by carbonyl reduction or methyl mono‐hydroxylation. A metabolic pathway involving N→O group reduction followed by acetyl group mono‐hydroxylation in only chicken was also proposed. There was also quantitative species difference for mequindox metabolism in three species. 1‐Desoxymequindox was the main metabolite in all species, but otherwise there were some qualitative interspecies differences in mequindox major metabolites. This work has revealed biotransformation characteristics of mequindox among different species, and moreover will further facilitate the explanations of the biological activities of mequindox in animals. Copyright © 2010 John Wiley & Sons, Ltd.