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Bacillus anthracis is an endospore-forming gram-positive pathogen that causes the infectious disease anthrax in mammals, including humans. Infections can occur via intradermal inoculation, ingestion, or inhalation of spores (24). Although anthrax infections via the former two routes are usually self-contained, inhalational anthrax is often lethal (23). In a mouse model of inhalational anthrax, inhaled B. anthracis spores are phagocytosed by alveolar macrophages that are believed to migrate to local lymph nodes (10). During migration, the spores germinate inside the macrophage phagolysosome to give rise to vegetative bacilli. The newly formed vegetative cells lyse the phagolysosome and replicate inside the macrophage cytoplasm (6), eventually escaping from the macrophage into the bloodstream. Therefore, in order to establish a successful anthrax infection, B. anthracis must survive and replicate intracellularly inside the macrophage, as well as extracellularly in the host's blood. Upon entering the bloodstream, B. anthracis is targeted by an array of innate immune mediators circulating in the host's blood, such as the complement proteins and cellular components such as neutrophils and platelets in humans. However, inhalational anthrax infection in animals is characterized by rapid progression into systemic bacteremia and the heavy growth of B. anthracis in the bloodstream (21). This observation indicates that B. anthracis is able not only to evade complement-mediated lysis and but also to resist the antibacterial activities of innate immune cells. One important antibacterial activity of innate immune cells in the human blood relies on the production of cationic antimicrobial peptides. These peptides are present in the cytosolic granules of neutrophils, eosinophils, and platelets and are released upon contact with bacterial pathogens (18). Cationic antimicrobial peptides interact electrostatically with negatively charged cell surface molecules, such as teichoic acids and phosphatidylglycerols of gram-positive bacteria, subsequently inducing disintegration of membrane structures and ultimately causing bacterial cell death (41). Some gram-positive pathogens, however, possess resistance mechanisms, by which they change cell surface properties and avoid killing by cationic antimicrobial peptides. For example, gram-positive pathogens, such as Staphylococcus aureus (29, 30), Listeria monocytogenes (1, 37), and Streptococcus pneumoniae (16), are able to be modify teichoic acids and phospholipids with d-alanine by DltABCD and l-lysine by MprF, respectively. Since these modifications contribute to a net positive charge on the cell surface, they are believed to facilitate repulsion of the cationic peptides. Identifying the B. anthracis genes that contribute to cationic peptide resistance can elucidate the molecular basis of this virulence trait. A recent study has shown that the B. anthracis genome contains a functional dltABCD operon (7). A B. anthracis mutant strain inactivated in this operon exhibits hypersusceptibility to various cationic antimicrobial peptides, decreased survival in macrophages, and virulence attenuation in a mouse model of inhalational infection. To date, the dltABCD operon is the only genetic determinant of B. anthracis experimentally proven to contribute to cationic antimicrobial peptide resistance. In the present study, we have identified a B. anthracis gene (BA1486 in the ΔANR [pXO1−, pXO2−] strain; BAS1375 in the Sterne 34F2 [pXO1+, pXO2−] strain) whose knockout leads to hypersusceptibility to protamine, as well as to human cationic antimicrobial peptides, α-helical LL-37, and β-sheet human neutrophil peptide 1 (HNP-1). We show that inactivation of this gene results in a strain that is unable to synthesize phosphatidylglycerols modified with lysine. Our results demonstrate that the B. anthracis genome carries a functional mprF gene required for cationic antimicrobial peptide resistance.