BACKGROUND: Cross-talk between skin keratinocytes (KCs) and Langerhans cells (LCs) plays a fundamental role in the body's first line of immunological defences. However, the mechanism behind the interaction between these two major epidermal cells is unknown. Interleukin (IL)-32 is produced in inflammatory skin disorders. We questioned the role of IL-32 in the epidermis. OBJECTIVES: We aimed to determine the role of IL-32 produced by KCs on surrounding LCs. METHODS: We used an ex vivo human explant model from healthy donors and investigated the role of IL-32 on LC activation using imaging, flow cytometry, reverse transcriptase quantitative polymerase chain reaction and small interfering (si)RNA treatment. RESULTS: Modified vaccinia virus ankara (MVA) infection induced KC death alongside the early production of the proinflammatory cytokine IL-32. We demonstrated that IL-32 produced by MVA-infected KCs induced modest but significant morphological changes in LCs and downregulation of adhesion molecules, such as epithelial cell adhesion molecule and very late antigen-4, and CXCL10 production. The treatment of KCs with IL-32-specific siRNA, and anti-IL-32 blocking antibody significantly inhibited LC activation, demonstrating the role of IL-32 in LC activation. We also found that some Toll-like receptor ligands induced a very high level of IL-32 production by KCs, which initiated LC activation. CONCLUSIONS: We propose, for the first time, that IL-32 is a molecular link between KCs and LCs in healthy skin, provoking LC migration from the epidermis to the dermis prior to their migration to the draining lymph nodes.