Objective The ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass/ mass spectrometry (UPLC-Q/ TOF-MS/ MS) urine metabolomics method was used to screen for potential metabolic markers of rat model of damp-heat diarrhea and to explore the pathogenesis. Methods The rat model of damp-heat diarrhea was established using a high-sugar and high-fat diet (HF), a high-temperature and high-humidity environment (HH), and intraperitoneal injection of Escherichia coli (EC). Urine samples were collected from rats at different stages of modeling (HF, HH, and EC) and analyzed using UPLC-Q/ TOF-MS/ MS. The data were analyzed by multivariate statistical analysis. Results Seven potential endogenous biomarkers were screened, namely, L-glutamic acid-L-cysteine, L-formyl kynurenine, 5-hydroxy-N-formylkynurenine, riboflavin-5-phosphate, linoleic acid, L-tryptophan and melatonin. Conclusions The metabolic pathways involved in damp heat diarrhea include metabolic pathways such as tryptophan metabolism, riboflavin metabolism, and linoleic acid metabolism. The results suggest that energy-, amino acid- and lipidmetabolism disorders occur in rats with damp-heat diarrhea which provide a scientific basis for elucidating the mechanism of pathogenesis of Chinese medicine and traditional Chinese veterinary medicine damp-heat diarrhea.