Type I interferons (IFNs), including IFN-β, are known to enhance antigen (Ag) presentation and to promote the expansion, survival and effector function of CD8+ cytotoxic lymphocytes (CTL) during viral infections. Furthermore, IFN-β is a potent candidate for antitumor drugs; however, recombinant IFN-β is too unstable for use in tumor therapy in vivo. In this study, we therefore examined the efficacy and mechanism of exogenous IFN-β as a biomolecule for tumor therapy, using adenovirus encoding IFN-β (Ad-IFNβ) as a therapeutic agent in a mouse model. Ag104Ld and 4T1 tumor cells exposed to Ad-IFNβ showed growth retardation and cell death in vitro, and tumor growth as well as tumor metastasis was inhibited in vivo. The Ad-IFNβ-mediated antitumor effect was dependent on CD8+ T cells in vivo, rather than on a direct cytotoxic effect of Ad-IFNβ. Transient T lymphocyte depletion was observed in tumor tissue after intratumoral injection with Ad-IFNβ. Despite the T lymphocyte depletion, the proliferation of Ag-specific CD8+ T cells was increased in Ad-IFNβ-treated mice compared to control virus-treated mice. These results suggest that IFN-β might contribute to the inhibition of tumor growth by depleting Ag-nonspecific T lymphocytes and enhancing proliferation of Ag-specific CD8+ T cells.