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.