Clostridium difficile has emerged as the important causative agent of antibiotics-associated pesudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis. We examined the pathophysiological roles of IFN-γ in toxin A-induced enteritis using IFN-γ knockout (KO) mice. When toxin A of C. difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection. IFN-γ protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice. On the contrary, at 2 and 4 h after toxin A injection, IFN-γ protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes. In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-α, macrophage inflammatory protein-1α and-2, KC, and ICAM-1> 2 h after treatment. In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-γ KO mice. Moreover, toxin A-induced gene expression of TNF-α, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-γ KO mice. Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-γ Ab prevented toxin A-induced enteritis. These observations indicate that IFN-γ is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-γ is an important molecular target for the control of C. difficile-associated pseudomembranous colitis.