In addition to its physical barrier against invading microorganisms, the skin produces antimicrobial peptides, human beta-defensins (hBDs) and cathelicidin LL-37, that participate in the innate host defense. Because IL-18 is produced by keratinocytes and involved in skin diseases in which hBDs and LL-37 are highly expressed, we hypothesized that these peptides would activate keratinocytes to secrete IL-18. We found that hBD-2, -3, and -4 and LL-37, but not hBD-1, activated normal human keratinocytes to secrete IL-18; this secretion reached peak strength at 3 h. In addition, the combination of peptides resulted in a synergistic effect on IL-18 secretion. We also revealed that hBD-2, -3, and -4 and LL-37 increased IL-18 mRNA expression, and that IL-18 secretion was more enhanced in keratinocytes differentiated in vitro with high Ca2+-containing medium. Furthermore, because IL-18 secretion induced by hBDs and LL-37 could not be suppressed by caspase-1 or caspase family inhibitors, and because these peptides failed to increase caspase-1 activity, we suggest that hBD- and LL-37-induced IL-18 secretion is probably via a caspase-1-independent pathway. To determine the molecular mechanism involved, we demonstrated that IL-18 secretion was through p38 and ERK1/2 MAPK pathways, because the inhibitors of p38 and ERK1/2, but not JNK, almost completely nullified IL-18 secretion. Moreover, hBD-2, -3, and -4 and LL-37 could induce the phosphorylation of p38 and ERK1/2, but not JNK. Thus, the ability of hBDs and LL-37 to induce IL-18 secretion by keratinocytes provides a new mechanism for these peptides in innate immunity and an understanding of their role in the pathogenesis of skin disorders.