Mammalian milk possesses inherent antimicrobial properties that have been attributed to several diverse molecules. Recently, antimicrobial peptides that belong to the cathelicidin gene family have been found to be important to the mammalian immune response. This antimicrobial is expressed in several tissues and increased in neonatal skin, possibly to compensate for an immature adaptive immune response. We hypothesized that the mammary gland could produce and secrete cathelicidin onto the epithelial surface and into milk. Human cathelicidin hCAP18/LL-37 mRNA was detected in human milk cells by PCR. Quantitative real-time PCR demonstrated an increase in relative expression levels at 30 and 60 d after parturition. Immunohistochemistry of mouse breast tissue identified the murine cathelicidin-related antimicrobial peptide in lobuloacinar and ductules. Western blot analysis of human milk showed that LL-37 was secreted and present in the mature peptide form. The antimicrobial activity of LL-37 against Staphylococcus aureus, group A Streptococcus, and enteroinvasive Escherichia coli O29 in the human milk ionic environment was confirmed by solution colony-forming assay using synthetic peptide. These results indicate that cathelicidin is secreted in mammary gland and human milk, has antimicrobial activity against both Gram-positive and Gram-negative bacteria, and can contribute to the anti-infectious properties of milk.