The incidence of ulcerative colitis (UC), an inflammatory disorder of the gastrointestinal tract, has rapidly increased in Asian countries over several decades. To overcome the limitations of conventional drug therapies, including biologics for UC management, the development of herbal medicine-derived products has received continuous attention. In this study, we evaluated the beneficial effects of a hydroethanolic extract ofFritillariae thunbergiiBulbus (FTB) in a mouse model of DSS-induced UC. The DSS treatment successfully induced severe colonic inflammation and ulceration. However, the severity of colitis was reduced by the oral administration of FTB. Histopathological examination showed that FTB alleviated the infiltration of inflammatory cells (e.g., neutrophils and macrophages), damage to epithelial and goblet cells in the colonic mucosal layer, and fibrotic lesions. Additionally, FTB markedly reduced the gene expression of proinflammatory cytokines and extracellular matrix remodeling. Immunohistochemical analysis showed that FTB alleviated the decrease in occludin and zonula occludens-1 expression induced by DSS. In a Caco-2 monolayer system, FTB treatment improved intestinal barrier permeability in a dose-dependent manner and increased tight junction expression. Overall, FTB has potential as a therapeutic agent through the improvement of tissue damage and inflammation severity through the modulation of intestinal barrier integrity.