ABSTRACT Clostridium difficile is a spore-forming bacterium that infects the lower intestinal tract of humans and is the most common known cause of diarrhea among hospitalized patients. Clostridium difficile colitis is mediated by toxins and develops during or following antibiotic administration. We have used a murine model of C. difficile infection, which reproduces the major features of the human disease, to study the effect of innate immune activation on resistance to C. difficile infection. We found that administration of purified Salmonella -derived flagellin, a Toll-like receptor 5 (TLR5) agonist, protects mice from C. difficile colitis by delaying C. difficile growth and toxin production in the colon and cecum. TLR5 stimulation significantly improves pathological changes in the cecum and colon of C. difficile -infected mice and reduces epithelial cell loss. Flagellin treatment reduces epithelial apoptosis in the large intestine, thereby protecting the integrity of the intestinal epithelial barrier during C. difficile infection. We demonstrate that restoring intestinal innate immune tone by TLR stimulation in antibiotic-treated mice ameliorates intestinal inflammation and prevents death from C. difficile colitis, potentially providing an approach to prevent C. difficile -induced pathology.