Immune imbalance plays a key role in the pathogenesis of ulcerative colitis (UC). The changes in CD4+ T cells and regulatory T cells (Tregs) in colonic T cells have an important relationship with UC. It is of great significance to elucidate the functional characteristics of CD4+ cells andTregs in UC patients. This study aimed to inquire into changes in functional markers of CD4+T cells and Tregs, including Helios, Bcl6, CTLA-4, CD226, TIGIT, PD-1 and ICOS. DSS-induced colitis was established in Balb/c mice and lymphocytes from spleen, mesenteric lymph nodes (MLNs), peripheral blood, and colon tissue were obtained. CD4+T cells and Tregs were analyzed by flow cytometry. We found that Helios+ and Bcl-6+ proportions were increased in colonic CD4+T cells of DSS-induced colitis mice. CD4+FoxP3+CXCR5-Tregs were significantly elevated in the colon of colitis mice, with CTLA-4+ percentages increased. Naïve subsets in colonic CD4+T and Tregs were significantly reduced, while effector T-cell subsets were increased in colitis mice. TIGIT+ and CD226+ percentages were significantly increased in both colonic CD4+T cells and Tregs of colitis mice. Both PD-1+ and ICOS+ percentages in colonic CD4+T cells and the ICOS+ percentage in colonic Tregs were significantly increased in colitis mice. In conclusion, functional molecules related to CD4+T cells and Tregs in colonic T cells are altered in UC, often adopting an activated phenotype. These changes may be associated with the pathogenesis of UC and could potentially serve as clinical therapeutic targets.