Urine is an important resource for biomarker research. Urine proteins reflect not only renal diseases but also changes in other organs in the body. However, urine has rarely been used to reflect on inflammatory bowel disease. In the present study, a trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model is used to mimic the human inflammatory bowel disease Crohn's disease (CD). Urine samples are analyzed by label-free and TMT-labeled proteomic quantitative methods. 77 urinary proteins are significantly changed in the colitis rats compared with that in the controls. These proteins are further validated by parallel reaction monitoring (PRM) targeted proteomic quantitative methods. Based on the human protein tissue atlas, carbonic anhydrase 1, ribonuclease pancreatic gamma type, and neutral and basic amino acid transport protein rBAT are highly enriched in the gastrointestinal tract. Among the nine PRM-validated proteins, carbonic anhydrase 1, neutrophil collagenase, and neutrophil gelatinase associated lipocalin were previously reported as IBD-associated proteins (all exhibiting consistent trends with the observation herein), whereas the others are newly discovered by this study. The results provide valuable clues for future study of urine biomarker of inflammatory bowel disease and Crohn's disease. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.