Precursors of α-defensin peptides require activation for bactericidal activity. In mouse small intestine, matrilysin colocalized with α-defensins (cryptdins) in Paneth cell granules, and in vitro it cleaved the pro segment from cryptdin precursors. Matrilysin-deficient (MAT^−/−) mice lacked mature cryptdins and accumulated precursor molecules. Intestinal peptide preparations from MAT^−/− mice had decreased antimicrobial activity. Orally administered bacteria survived in greater numbers and were more virulent in MAT^−/− mice than in MAT^+/+ mice. Thus, matrilysin functions in intestinal mucosal defense by regulating the activity of defensins, which may be a common role for this metalloproteinase in its numerous epithelial sites of expression.