Direct in vivo administration of a lentiviral vaccine has been shown to transduce dendritic cells (DCs) in order to induce antigen-specific CD8+ T cell responses, but the efficacy of antitumor immunity has not been reported. In this study we tested whether direct in vivo administration of a lentiviral vaccine can induce selfantigen- based therapeutic antitumor immunity in murine tumor models. Lentiviral vector (LV) transduced DCs efficiently in vitro and was able to transduce DCs in vivo. LV-transduced DCs effectively presented antigens to T cells. Compared with a naked DNA tyrosinase-related protein-2 (TRP2)-heat shock protein-70 (hsp70) vaccine, the TRP2-specific interferon-gamma-producing CD8+ T cell response was augmented by direct in vivo administration of an LV-TRP2hsp70 vaccine, which induced significant therapeutic antitumor immunity in subcutaneous B16 and subcutaneous GL-26 models. Moreover, in vivo administration of an LV-NeuEDhsp70 vaccine induced significant therapeutic antitumor immunity against spontaneous breast tumors in a BALB/c- Neu transgenic model. Our observations indicate that direct in vivo administration of a lentiviral vaccine not only enhances antigen-specific CD8+ T cell responses, but also generates significant therapeutic antitumor activities.