Background <p>Differentiation between ulcerative colitis (UC) and other intestinal inflammatory diseases is difficult, and the precise etiology of UC is poorly understood. Thus, there is a need for novel diagnostic and prognostic biomarkers for UC.</p> Methods <p>Intestinal mucosal biopsy tissue specimens of inflamed (ulcerative colitis-inflamed, UC-I) and non-inflamed (ulcerative colitis-noninflamed, UC-N) tissue were obtained simultaneously during colonoscopy from newly diagnosed UC patients prior to any treatments. Label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) quantitative proteomics was used to detect proteomic differences between UC-I, UC-N, and normal control subjects (<i>n</i> = 5). Proteins with a fold-change &gt; 1.5 and <i>P</i> &lt; 0.05 between groups were considered to be differentially expressed (DEPs). Candidate biomarkers were further verified in 8 patients of each group by parallel reaction monitoring (PRM) (a prospective cohort, <i>n</i> = 8). Expression of TXNDC5 was quantified using immunohistochemistry (IHC).</p> Results <p>A total of 4,788 proteins were identified. Multiple upregulated pathways, including leukocyte trans-endothelial migration and natural killer (NK) cell-mediated cytotoxicity, were identified. Network analysis showed that proteins were involved in 4 pathways in UC-I and 3 pathways in UC-N tissues, and participated in protein-protein interactions. Increased expression of 9 DEPs, including TXNDC5, EPX, and ITGAM were detected in UC patients compared to normal control subjects. Subsequent verification of the 9 DEPs by PRM confirmed the reliability of the mass spectrometry data. TXNDC5 expression was significantly increased in UC.</p> Conclusions <p>The pathways, networks, and proteins identified in this study may provide new insights into the molecular pathogenesis of UC. Further studies are required to determine if the proteins identified may help in the diagnosis and treatment of UC.</p>