Carcinoembryonic antigen (CEA) is expressed on human colon carcinomas, is well characterized, and continues to be a promising target for cancer immunotherapy in humans. To enhance the immunogenecity of CEA, we developed a fusion gene (CRT-TAT-DeltaCEA) of the TAT protein transduction domain (PTD) and calreticulin (CRT) with human CEA devoid of its signal sequences (DeltaCEA) and evaluated anti-tumor immunity using RNA-pulsed dendritic cell (DC) vaccination. Mice vaccinated with DC by electroporation with mRNA encoding TAT-DeltaCEA (DC/TAT-DeltaCEA) and CRT-DeltaCEA (DC/CRT-DeltaCEA) had enhanced induction of tumor-specific cytotoxic T lymphocyte (CTL) and increased numbers of IFN-gamma-secreting T cells by ELISPOT, as compared to mice vaccinated with DC/DeltaCEA. DC/CRT-DeltaCEA and DC/TAT-DeltaCEA vaccines preferentially stimulated CD4+ and CD8+ T cells, respectively. The DC vaccine by electroporation with mRNA encoding CRT-TAT-DeltaCEA (DC/CRT-TAT-DeltaCEA) enhanced both CD4+ and CD8+ T cells. DC/CRT-TAT-DeltaCEA had the additional effects of CRT and TAT PTD and enhanced the anti-tumor effect against CEA-expressing tumors compared to DC/CRT-DeltaCEA or DC/TAT-DeltaCEA. These findings suggest that modification of CEA with both CRT and TAT PTD induces potent anti-tumor immune responses in RNA-pulsed DC vaccination and may be a useful approach for DC-based immunotherapy.