To investigate roles of IL-23 in viral infection, we have engineered recombinant vaccinia virus (VV) expressing IL-12 (VV-IL-12) and expressing IL-23 (VV-IL-23). We found VV-IL-23 was less virulent in BALB/c mice than wild-type VV (VV-WT), indicating that IL-23 enhances resistance to VV. VV-specific CTL activity in VV-IL-23-infected mice was slightly higher than activity in VV-WT-inoculated mice, although antiviral Ab production and NK activity were not increased. IL-12/23p40-deficient mice survived the infection with VV-IL-23, indicating that IL-23 promotes VV resistance independently of IL-12. The mechanism of the IL-23-mediated resistance was distinct from that of the IL-12-regulated resistance because IFN-γ-deficient mice did not eliminate VV-IL-12, but did eradicate VV-IL-23. These data indicate that IFN-γ is essential for the IL-12-mediated resistance, but dispensable for the IL-23-regulated resistance. Because IL-17 is a key in the IL-23-regulated resistance to bacteria, we hypothesized an involvement of IL-17 in the resistance to VV. Treatment with an anti-IL-17 mAb resulted in a significant increase of viral titers in VV-IL-23-infected IFN-γ-deficient mice. In addition, VV-IL-17 was less virulent than VV-WT in BALB/c mice, and IL-17-deficient mice were more sensitive to VV-WT than control mice. However, the effect of neutralization with an anti-IL-17 mAb was limited, and IL-17-deficient mice survived the infection with VV-IL-23. Taken together, these data suggest that the IL-23/IL-17 axis plays a certain but subdominant role in the IL-23-mediated resistance to VV. Unveiling of an alternative pathway in the IL-23-regulated resistance might provide a novel strategy against infectious pathogens without side effects of autoimmunity.