A therapeutically effective cancer vaccine must generate potent antitumor immune responses and be able to overcome tolerance mechanisms mediated by the progressing tumor itself. Previous studies showed that glycoprotein 100 (gp100), tyrosinase-related protein 1 (TRP1), and tyrosinase-related protein 2 (TRP2) are promising immunogens for melanoma immunotherapy. In this study, we administered these three melanoma-associated antigens via lentiviral vectors (termed LV-3Ag) and found that this multi-antigen vaccine strategy markedly increased functional T-cell infiltration into tumors and generated protective and therapeutic antitumor immunity. We also engineered a novel immunotoxin, αFAP-PE38, capable of targeting fibroblast activation protein (FAP)-expressing fibroblasts within the tumor stroma. When combined with αFAP-PE38, LV-3Ag exhibited greatly enhanced antitumor effects on tumor growth in an established B16 melanoma model. The mechanism of action underlying this combination treatment likely modulates the immune suppressive tumor microenvironment and, consequently, activates cytotoxic CD8(+) T cells capable of specifically recognizing and destroying tumor cells. Taken together, these results provide a strong rationale for combining an immunotoxin with cancer vaccines for the treatment of patients with advanced cancer.