Interleukin (IL-) 27 is a member of IL-12 cytokine family with Th1-promoting and anti-inflammatory effects. IL-27 has been shown to facilitate tumor-specific cytotoxic T lymphocyte (CTL) induction against various tumors. However, IL-27 suppresses cytokine production of lymphocytes and antigen-presenting function of dendritic cells (DCs). To examine the in vivo role of IL-27 in generation of anti-tumor immunity, we examined IL-27-mediated antitumor-effects using WSX-1 (IL-27 receptor α chain)-deficient (WSX-1−/−) mice. In WSX-1−/− mice inoculated with B16 melanoma cells, tumor growth was higher than in wild-type (WT) mice. Accordingly, tumor-specific CTL generation was lower in WSX-1−/− mice than in WT mice. CTL induction in WSX-1−/− mice was not restored by transfer of WT DCs pulsed with TRP2 peptide, indicating that IL-27 is directly required for generation of tumor-specific CTLs. However, when transferred into tumor-bearing mice, WSX-1−/− DCs pulsed with TRP2 peptide was more potent than WT DCs in tumor growth inhibition and generation of CTLs, indicating suppressive effects of IL-27 on DC function. Finally, the combination of WT CD8+ T cells and KO DCs is more potent in generation of antigen-specific CTLs than any other combinations. Expression of perforin gene and percentages of tumor-specific CD8+ T cells were also the highest in the combination of WT CD8+ T cells and WSX-1−/− DCs. It was thus revealed that IL-27 promotes CTL generation while suppressing DC function during generation of tumor immunity. The combination of WT T cells and IL-27 signal-defective DCs may have therapeutic potential against tumors. © 2008 Wiley-Liss, Inc.