Fractalkine (CX3CL1) is a unique membrane-bound CX3C chemokine that serves as a potent chemoattractant for lymphocytes. The hypothesis of this study is that dendritic cells (DC) genetically modified ex vivo to overexpress fractalkine would enhance the T cell-mediated cellular immune response with a consequent induction of anti-tumor immunity to suppress tumor growth. To prove this hypothesis, established tumors of different mouse cancer cells (B16-F10 melanoma, H-2b, and Colon-26 colon adenocarcinoma, H-2d) were treated with intratumoral injection of bone marrow-derived DC that had been modified in vitro with an RGD fiber-mutant adenovirus vector expressing mouse fractalkine (Ad-FKN). In both tumor models tested, treatment of tumor-bearing mice with Ad-FKN-transduced DC gave rise to a significant suppression of tumor growth along with survival advantages in the treated mice. Immunohistochemical analysis of tumors treated with direct injection of Ad-FKN-transduced DC demonstrated that the treatment prompted CD8+ T cells and CD4+ T cells to accumulate in the tumor milieu, leading to activation of immune-relevant processes. Consistent with the finding, the intratumoral administration of Ad-FKN-transduced DC evoked tumor-specific cytotoxic T lymphocytes, which ensued from in vivo priming of Th1 immune responses in the treated host. In addition, the anti-tumor effect provided by intratumoral injection of Ad-FKN-transduced DC was completely abrogated in CD4+ T cell-deficient mice as well as in CD8+ T cell-deficient mice. These results support the concept that genetic modification of DC with a recombinant fractalkine adenovirus vector may be a useful strategy for cancer immunotherapy protocols.