Although tumors express potentially immunogenic tumor-associated antigens (TAAs), cancer vaccines often fail because of inadequate antigen delivery and/or insufficient activation of innate immunity. Engineering nonpathogenic bacterial vectors to deliver TAAs of choice may provide an efficient way of presenting TAAs in an immunogenic form. In this study, we used genes of Salmonella pathogenicity island 2 (SPI2) to construct a novel cancer vaccine in which a TAA, survivin, was fused to SseF effector protein and placed under control of SsrB, the central regulator of SPI2 gene expression. This construct uses the type III secretion system (T3SS) of Salmonella and allows preferential delivery of tumor antigen into the cytosol of antigen-presenting cells for optimal immunogenicity. In a screen of a panel of attenuated strains of Salmonella, we found that a double attenuated strain of Salmonella typhimurium, MvP728 (purD/htrA), was not toxic to mice and effectively expressed and translocated survivin protein inside the cytosol of murine macrophages. We also found that a ligand for CD1d-reactive natural killer T (NKT) cells, alpha-glucuronosylceramide (GSL1), enhanced MvP728-induced interleukin-12 production in human dendritic cells and that in vivo coadministration of a NKT ligand with MvP728-Llo or MvP728-survivin enhanced effector-memory cytotoxic T lymphocyte (CTL) responses. Furthermore, combined use of MvP728-survivin with GSL1 produced antitumor activity in mouse models of CT26 colon carcinoma and orthotopic DBT glioblastoma. Therefore, the use of TAA delivery via SPI-2-regulated T3SS of Salmonella and NKT ligands as adjuvants may provide a foundation for new cancer vaccines.