The purpose of this study was to investigate the effect of radiation on the content of animal diet constituents using global metabolomics. Aqueous methanolic extracts of control and cobalt-60-irradiated Teklad 7001 diets were comprehensively analyzed using nano-liquid chromatography–MS/MS. Among the over 2000 ions revealed by XCMS followed by data preprocessing, 94 positive and 143 negative metabolite ions had greater than 1.5-fold changes and p-values <0.01. Use of MetaboAnalyst statistical software demonstrated complete separation of the irradiated and non-radiated diets in unsupervised principal components analysis and supervised partial least squares discriminant analysis. Irradiation led to an increase in the content of phytochemicals such as glucosinolates and oxidized lipids in the diet. Twenty-eight metabolites that were significantly changed in the irradiated samples were putatively identified at the level of molecular formulae by MS/MS. MS/MSALL analysis of chloroform–methanol extracts of the irradiated diet showed increased levels of a number of unique linoleic acid-derived branched fatty acid esters of hydroxy fatty acids. These data imply that gamma irradiation of animal diets causes chemical changes to dietary components, which in turn may influence the risk of mammary cancer. Copyright © 2017 John Wiley & Sons, Ltd.