Interleukin (IL)-33 is a cytokine belonging to the IL-1 family. IL-33 plays an important role in Th2 immune responses, and induces goblet cell hyperplasia in the intestinal mucosa. In this study, to elucidate the molecular mechanisms underlying IL-33-induced goblet cell hyperplasia, we investigated how IL-33 modulates the Notch signaling pathway in dextran sulfate sodium (DSS)-induced experimental colitis. DSS colitis was induced in BALB/c mice with intraperitoneal administrations of IL-33 (1 µg/body) every 48 h. Tissue samples were evaluated by standard immunohistochemical procedures. The mucosal mRNA expression of the Notch ligands was analyzed by a real-time polymerase chain reaction. The mucosal mRNA expression of Notch ligands [Jagged1 (Jag1) and Delta-like (Dll) 1 and 4] was significantly increased in DSS-colitis mice. IL-33-induced goblet cell hyperplasia in the control mice. In the DSS-colitis mice, the goblet cells were depleted in the colon, but IL-33 completely prevented goblet cell depletion in the DSS-colitis mice. IL-33 induced a significant decrease in Jag1 and Dll4 mRNA expression in the mucosa of the control mice. Mucosal mRNA expression for Jag1, Dll1 and 4 was significantly elevated in the DSS-colitis mice, but this elevation was significantly blocked by the administration of IL-33. IL-33 dose-dependently decreased Jag1 mRNA expression in mouse colonic subepithelial myofibroblasts. In contrast to its preventive effects on goblet cell depletion, IL-33 aggravated DSS colitis. IL-33 prevented goblet cell depletion via its inhibitory actions against Notch ligand expression in DSS colitis, but exacerbated the disease activity. IL-33 plays two counter actions in mucosal inflammation; the first is a protective action via goblet cell induction, and the second is a pro-inflammatory action as a Th2 cytokine.