Objective? The aim of the study was to investigate the expression of proteins in colonic tissues of mice with ulcerative colitis (UC) by using isobaric tags for relative and absolute quantitation (iTRAQ), probe into the pathogenesis of UC, and find potential biomarkers of UC. Methods? Forty C57 mice were randomly divided into the control and model groups (20 mice in each group). The mice in the model group were administered dextran sulphate sodium (DSS) for 7 consecutive days ad libitum to induce acute colitis, and the colon tissue was extracted on the 8th day after the successful establishment of the UC model. Proteins were identified by the iTRAQ and tandem mass spectrometry techniques,and the identified proteins were analyzed by bioinformatics. Results? A total of 4019 proteins were identified among the two groups. Among them, 317 significant differentially expressed proteins (DEPs) were detected according to the screening criteria for selecting DEPs, i.e. fold change ratios ≥ 1.5 or ≤ 0.67 and P-values < 0.05, of which 156 were upregulated and 161 were downregulated. In the Gene Ontology (GO) analysis, the DEPs were classified into 48 functional categories, which contained biological process, cellular component, and molecular function. Based on the 317 DEPs, the KEGG pathway analysis identified 160 vital pathways. Conclusion? DEPs in colonic tissues of mice with UC were screened using the iTRAQ technique, which laid a foundation for further studies regarding the pathogenesis of UC.