Lung cysteine cathepsins B, K, L, and S contribute to physiological and pathological processes including degradation of antimicrobial peptides/proteins (AMPs) such as surfactant protein SP-A, lactoferrin, secretory leukocyte peptidase inhibitor, and beta-defensins-2 and -3. Substantial amounts of uncleaved LL-37, a 37-mer cationic AMP, were observed in the sputum of patients with cystic fibrosis (CF). Nevertheless LL-37 was degraded after prolonged incubation in CF sputum, and the hydrolysis was blocked by E-64, a selective inhibitor of cysteine proteases. Cathepsins K and S, expressed in human alveolar macrophages, thoroughly hydrolyzed LL-37 in vitro, whereas it competitively inhibited cathepsin L (Ki = 150 nM). Cleavage of LL-37 by cathepsins S and K impaired its antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus, in a time- and concentration-dependent manner. The exchange of residues 67 and 205 in the S2 pockets of cathepsins L (Leu67Tyr/Ala205Leu) and K (Tyr67Leu/Leu205Ala) switched the specificity of these mutants toward LL-37. Molecular modeling suggested that LL-37 interacted with the active site of cathepsin L in both forward (i.e., substrate-like) and reverse orientations with similar binding energies. Our data support the hypothesis that cysteine cathepsins modulate the innate immunity response by degrading distinct and representative members of the AMP family.