Postoperative fatigue syndrome (POFS) is a common clinical complication followed by almost every major abdominal surgery. There is not a full explanation to the etiology of POFS, especially its central mechanism. Carthamus tinctorius L is a classic traditional Chinese medicine (TCM) which could exert the anti-fatigue effect on POFS. However, its mechanism is still lacking. Here, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS) based metabonomic approach was used to characterize hippocampal metabolic fluctuations of POFS in a rat model induced by partial hepatectomy (PHx), and to evaluate the anti-fatigue effect of Carthamus tinctorius L extract (CTLE). With partial least-squares discriminant analysis for classification and selection of biomarkers, fifteen hippocampal metabolites related to POFS were identified, primarily involving alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine degradation, purine metabolism, phenylalanine metabolism, tryptophan metabolism, phospholipid metabolism and fatty acid metabolism. With these altered metabolic pathways as possible drug targets, we systematically analyzed the protective effect of CTLE, which showed that CTLE could provide anti-fatigue effect on POFS through partially regulating the perturbed metabolic pathways. This study indicated that UHPLC-Q-TOFMS-based metabolomics provided a powerful tool to reveal hippocampal metabolic fluctuations of POFS and study the mechanism of TCM. This article is protected by copyright. All rights reserved.