Semliki Forest virus (SFV) represents a promising gene therapy vector for tumor treatment, because it produces high levels of recombinant therapeutic proteins while inducing apoptosis in infected cells. In this study, we constructed a SFV vector expressing murine interferon alpha (IFNα). IFNα displays antitumor activity mainly by enhancing an antitumor immune response, as well as by a direct antiproliferative effect. In spite of the antiviral activity of IFNα, SFV-IFN could be produced in BHK cells at high titers. This vector was able to infect TC-1 cells, a tumor cell line expressing E6 and E7 proteins of human papillomavirus, leading to high production of IFNα both in vitro and in vivo. When injected into subcutaneous TC-1 tumors implanted in mice, SFV-IFN was able to induce an E7-specific cytotoxic T lymphocyte response, and to modify tumor infiltrating immune cells, reducing the percentage of T regulatory cells and activating myeloid cells. As a consequence, SFV-IFN was able to eradicate 58% of established tumors treated 21 days after implantation with long-term tumor-free survival and very low toxicity. SFV-IFN was also able to induce significant antitumor responses in a subcutaneous tumor model of murine colon adenocarcimoma. These data suggest that local production of IFNα by intratumoral injection of recombinant SFV-IFN could represent a potent new strategy to treat tumors in patients.