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‐2^b, and Colon‐26 colon adenocarcinoma, H‐2^d) 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.