Background: Ulcerative colitis (UC) is a lifelong chronic inflammatory disease that is characterized by the absence of specific markers for diagnosis and prognosis. TPM3 is an integral component of the thin filament, responsible for the structural stability of actin filaments and modulation of cytoskeletal function. This study investigated the regulatory role of TPM3 in UC and its potential mechanisms. Methods: At the clinical level, TPM3 levels were assessed in serum and mucosal tissues of UC and other enteric disease. At the cellular level, the effects of TMP3 overexpressing lentivirus on Caco-2 cell phenotype and the barrier of IL-1 beta-induced UC model were explored. At the animal level, the effects of TMP3 overexpressing lentivirus on symptoms and colonic damage in a DSS-induced UC model were explored. Results: TPM3 expression in serum of UC patients was significantly lower than that of other enteric disease, and TPM3 levels in the intestinal mucosa showed a negative correlation with the Mayo score of UC patients. TPM3 overexpression alleviates IL-1 beta-induced apoptosis and inhibition of invasion and migration in UC model in vitro. In monolayer Caco-2 cells, TPM3 overexpression rescued the IL-1 beta-induced decrease in transepithelial electrical resistance and tight junction markers (ZO-1 and Occludin) and increase in permeability. In animal experiments, TPM3 overexpression increased body weight and colon length and decreased disease activity index in a DSSinduced UC model. In tissue staining, it alleviated pathological damage and upregulated Occuludin and TPM3 levels in the colon. Conclusion: TPM3 levels correlated with UC disease course and TPM3 overexpression alleviated symptoms/ phenotypes and barrier damage in UC models in vivo and in vitro. TPM3 may serve as a potential novel biomarker for UC diagnosis and prognosis.