Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) agonist, was found to exacerbate inflammation and tissue injury in experimental acute colitis mice. Through lipidomics analysis, bioactive sphingolipids were significantly up-regulated in the colitis group. In this study, to provide further insight into the PPARα-dependent exacerbation of colitis, gas chromatography-mass spectrometry (GC/MS) based metabolomics was employed to investigate the serum and colon of dextran sulfate sodium (DSS)-induced colitis mice treated with fenofibrate, with particular emphasis on changes in low-molecular-weight metabolites. With the aid of multivariate analysis and metabolic pathway analysis, potential metabolite markers in the amino acid metabolism, urea cycle, purine metabolism, and citrate cycle were highlighted, such as glycine, serine, threonine, malic acid, isocitric acid, uric acid, and urea. The level changes of these metabolites in either serum or colons of colitis mice were further potentiated following fenofibrate treatment. Accordingly, the expression of threonine aldolase and phosphoserine aminotransferase 1 was significantly up-regulated in colitis mice and further potentiated in fenofibrate/DSS-treated mice. It was revealed that beyond the control of lipid metabolism, PPARα also shows effects on the above pathways, resulting in enhanced protein catabolism and energy expenditure, increased bioactive sphingolipid metabolism and proinflammatory state, which were possibly related to the exacerbated colitis.