Recent studies have shown that IL-18, a pleiotropic cytokine that augments IFN-γ production, is produced by intestinal epithelial cells and lamina propria cells from patients with Crohn’s disease. In this study, we show that IL-18 is strongly expressed by intestinal epithelial cells in a murine model of Crohn’s disease induced by transfer of CD62L+ CD4+ T cells into SCID mice. To specifically down-regulate IL-18 expression in this model, we constructed an E1/E3-deleted adenovirus expressing IL-18 antisense mRNA, denoted Ad-asIL-18, and demonstrated the capacity of such a vector to down-regulate IL-18 expression in colon-derived DLD-1 cells and RAW264. 7 macrophages. Local administration of the Ad-asIL-18 vector to SCID mice with established colitis led to transduction of epithelial cells and caused a significant suppression of colitis activity, as assessed by a newly developed endoscopic analysis system for colitis. Furthermore, treatment with Ad-asIL-18 induced a significant suppression of histologic colitis activity and caused suppression of mucosal IFN-γ production, whereas IFN-γ production by spleen T cells was unaffected. Taken together, these data indicate an important role for IL-18 in the effector phase of a T cell-dependent murine model of colitis and suggest that strategies targeting IL-18 expression may be used for the treatment of patients with Crohn’s disease.