Evidences suggest that dietary docosahexaenoic acid (DHA) supplementation may have pleiotropic beneficial effects on health. However, the underlying mechanisms and crucial targets that are involved in achieving these benefits remain to be clarified. In this study, we employed biochemical analysis and liquid chromatography-mass spectrometry (LC-MS) based untargeted metabolomics coupled with multivariate statistical analysis to identify potential metabolic targets of DHA in adult rats at 48 h postfeeding. Blood biochemical analysis showed a significant decrease in triglyceride level of DHA diet group, the untargeted metabolomic analysis revealed that some metabolites were significantly different between the DHA diet group and the basal diet group, including fatty acids (16:0, 18:1, 20:5n3, 22:2n6 and 24:0), diglyceride (20:0/18:2n6, 18:3n6/22:6n3, 20:4n3/20:4n3, and 22:0/24:0), PIP2 (18:2/20:3), phytol, lysoSM (d18:1), 12-hydroxyheptadecatrienoic acid, dihydrocorticosterone and N-1- acetylspermine, which are mainly involved in fat mobilization and triglyceride hydrolysis, arachidonic acid, steroid hormone, and polyamine metabolism. To our knowledge, this is the first report that links the metabolic effects of DHA with arachidonic acid, steroid, and polyamine metabolism. Our finding suggests that the beneficial effects of DHA, may not directly require its own metabolic derivatives, but could be achieved by metabolic regulation. (C) 2020 Elsevier Inc. All rights reserved.