Interleukin (IL)-27 is a member of IL-12 family cytokine. We have previously reported that IL-27 inhibits human immunodeficiency virus type-1 (HIV-1) replication in CD4(+) T cells and monocyte-derived macrophages, even though IL-12 enhances HIV-1 replication in primary CD4(+) T cells. Further study demonstrates that IL-27 induces antiviral genes including RNA-dependent protein kinase, oligoadenylate synthetase, and myxovirus protein in the same manner as interferon (IFN)-alpha. Neutralization assay using anti-IFN antibodies, real-time RT-PCR, and enzyme-linked immunosorbent assay demonstrated that IL-27 induces the antiviral genes without the induction of IFNs. IFN-alpha has been administered to hepatitis C virus (HCV)-infected patients as well as HCV/HIV-1 co-infected patients. Despite the improved immunotherapy, some patients are still failed to respond to the treatment. Since IL-27 induces IFN-alpha-like responses including the induction of antiviral genes, it was speculated that IL-27 may impact the replication of HCV. In this study, we evaluated the role of IL-27 on HCV replication using Huh7.5, an HCV permissive cell line. IL-27 induces STAT-1 and -3 in the cell line, and dose-dependently inhibited HCV. These data suggest that IL-27 may play a role in the development of a novel immunotherapeutic strategy for HCV and HCV/HIV co-infection.