Clostridium difficile, the causative agent of antibiotic-associated colitis, mediates inflammatory diarrhea by releasing toxin A, a potent 308-kDa enterotoxin. Toxin A-induced inflammatory diarrhea involves many steps, including mucosal release of substance P (SP) corticotropin-releasing hormone (CRH) and neutrophil transmigration. Here we demonstrate that, compared with wild type, mice genetically deficient in CRH (Crh(-/-)) have dramatically reduced ileal fluid secretion, epithelial cell damage, and neutrophil transmigration 4 h after intraluminal toxin A administration. This response is associated with diminished mucosal activity of the neutrophil enzyme myeloperoxidase compared with that of wildtype mice. In wild-type mice, toxin A stimulates an increase in intestinal SP content compared with buffer administration. In contrast, toxin A administration in Crh(-/-) mice fails to result in an increased SP content. Moreover, immunohistochemical experiments showed that CRH and SP are colocalized in some enteric nerves of wild-type mice, and this colocalization is more evident after toxin A administration. These results provide direct evidence for a major proinflammatory role for CRH in the pathophysiology of enterotoxin-mediated inflammatory diarrhea and indicate a SP-linked pathway.