The presence of cathelicidin antimicrobial peptides provides an important mechanism for prevention of infection against a wide variety of microbial pathogens. The activity of cathelicidin is controlled by enzymatic processing of the proform (hCAP18 in humans) to a mature peptide (LL-37 in human neutrophils). In this study, elements important to the processing of cathelicidin in the skin were examined. Unique cathelicidin peptides distinct from LL-37 were identified in normal skin. Through the use of selective inhibitors, SELDI-TOF-MS, Western blot, and siRNA, the serine proteases stratum corneum tryptic enzyme (SCTE, kallikrein 5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7) were shown to control activation of the human cathelicidin precursor protein hCAP18 and also influence further processing to smaller peptides with alternate biological activity. The importance of this serine protease activity to antimicrobial activity in vivo was illustrated in SPINK5-deficient mice that lack the serine protease inhibitor LEKTI. Epidermal extracts of these animals show a significant increase in antimicrobial activity compared with controls, and immunoabsorption of cathelicidin diminished antimicrobial activity. These observations demonstrate that the balance of proteolytic activity at an epithelial interface will control innate immune defense.