Cathelicidin-related antimicrobial peptide (mCRAMP), the sole murine cathelicidin, is encoded by the gene Cnlp. We show that mCRAMP expression in the intestinal tract is largely restricted to surface epithelial cells in the colon. Synthetic mCRAMP had antimicrobial activity against the murine enteric pathogen Citrobacter rodentium, which like the related clinically important human pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli, adheres to the apical membrane of intestinal epithelial cells. Colon epithelial cell extracts from Cnlp+/+ mice had significantly greater antimicrobial activity against C. rodentium than those of mutant Cnlp−/− mice that lack mCRAMP. Cnlp−/− mice developed significantly greater colon surface and crypt epithelial cell colonization, surface epithelial cell damage, and systemic dissemination of infection than Cnlp+/+ mice after oral infection with C. rodentium. Moreover, Cnlp+/+ mice were protected from oral infections with C. rodentium inocula that infected the majority of Cnlp−/− mice. These results establish cathelicidin as an important component of innate antimicrobial defense in the colon.