Type I interferons (IFN) have an essential role in antiviral defense, and they are produced upon viral infection in a variety of cells. IFN-alpha/beta treatment of immature dendritic cells (DC) is known to induce their phenotypic and functional maturation, but it remains unclear whether stimulation by this cytokine family influences the functions and maturation of Langerhans cells (LC). We used highly enriched (>95%) LC directly isolated from BALB/c mouse skin and addressed this issue, comparing LC with splenic CD11c(+) DC. Type I IFN-treated LC exhibited impaired ability to produce IL-12 and inflammatory cytokines, IL-6 and TNF-alpha, whereas IL-10 production was not augmented. In splenic DC, the production of inflammatory cytokines was rather enhanced by type I IFN treatment. With regard to chemokines, in both LC and splenic DC, type I IFN upregulated the production of inflammatory chemokines, such as CXCL10, CXCL11, CCL3, CCL4, and CCL5. Strikingly, IFN-beta treatment reduced the expression of CD40, CD54, CD80, and CD86 on LC, whereas IFN-beta-treated splenic DC showed enhanced expression of these molecules. Furthermore, IFN-beta-treated LC had impaired costimulatory activity for anti-CD3-induced proliferation of T cells. Finally, treatment with IFN-alpha/beta reduced the migratory capacity of LC to CCL21. These results indicate that type I IFN inhibit maturation and activation of LC in a direct manner. Our observations may provide a novel explanation for the reported inability of LC to act as potent antigen-presenting cells in cutaneous and mucosal viral infection.