Cathelicidins are a class of small cationic peptide antibiotics that are expressed in skin and in other epithelial cells and are an active component of mammalian innate immunity. Human cathelicidin (hCAP18/LL-37) consists of a conserved prosequence called the cathelin-like domain and a C-terminal peptide named LL-37. To date, our understanding of the cathelin-like domain was very limited. To bring insight into the function of this evolutionarily conserved prosequence, we produced recombinant human cathelin-like protein and full-length hCAP18/LL-37 in Escherichia coli. As the cathelin-like protein shares homology with the cystatin family of cysteine protease inhibitors, we first analyzed the effect of the cathelin-like recombinant protein on the cysteine protease cathepsin L. We found that the cathelin-like protein inhibited protease activity. Next, we tested the cathelin-like protein for antimicrobial activity using solid phase radial diffusion and liquid phase killing assays. The cathelin-like prosequence, but not full-length hCAP18/LL-37, killed human pathogens including E. coli and methicillin-resistant Staphylococcus aureus at concentrations ranging from 16 to 32 microM. Together these findings suggest that after proteolytic cleavage the cathelin-like domain can contribute to innate host defense through inhibition of bacterial growth and limitation of cysteine-proteinase-mediated tissue damage. As these dual functions are complementary to the LL-37 peptide released from the C-terminus of full-length hCAP18/LL-37, human cathelicidin represents an elegant multifunctional effector molecule for innate immune defense of the skin.