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Plantaginis semen, the dried mature seed of Plantago asiatica L. or Plantago deprdssa Willd., has a prominent effect on the treatment of obesity, type 2 diabetes and lipid disorders, however, its clinical application is limited due to inadequate in-depth mechanism exploration and incomplete discussion of action targets of its in vivo. Therefore, an untargeted metabolomics approach was firstly applied to study the serum metabolic differences in mice. Metabolomics analysis was performed using ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) together with multivariate statistical data analysis. The results showed that Plantaginis semen can mainly improve blood lipids, some degree in blood glucose and insulin levels in high-fat mice, in addition, the phenotype of liver and fat stained sections demonstrated remarkable results. A total of 22 metabolites involved in arachidonic acid, glycerophospholipid, glycosphingolipid, linoleate, Omega-3 fatty acid, phosphatidylinositol phosphate and tyrosine metabolisms were identified. In further, it was found that the possible mechanisms of Plantaginis semen on hyperlipidemic mice lied in the biosynthesis of thyroxine, biological effects of enzymes of phospholipase A2 activity, glucosylceramide synthase and inositol essential enzyme 1α, genes expressions of fatty acid metabolism and inflammation. Serum metabolomics revealed that Plantaginis semen could cure the organism disease via regulating multiple metabolic pathways which will be helpful for understanding the mechanism of this herb and providing references for better applications of it in clinic, even researches on other TCMs., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)