Innate immunity serves as a first line defense in vertebrate organisms by providing an initial barrier to microorganisms and triggering antigen-specific responses. Antimicrobial peptides are thought to be effectors of innate immunity through their antibiotic activity and direct killing of microorganisms. Evidence to support this hypothesis in vertebrates is indirect, based on expression profiles and in vitro assays using purified peptides. Here we investigated the function of antimicrobial peptides in vivo using mice deficient in an antimicrobial peptide, mouse β-defensin-1 (mBD-1). We find that loss of mBD-1 results in delayed clearance of Haemophilus influenzae from lung. These data demonstrate directly that antimicrobial peptides of vertebrates provide an initial block to bacteria at epithelial surfaces.