Heat-shock protein (HSP)-based immunotherapy is established on its adjuvant effects when applied via an extracellular approach to pulse and activate dendritic cells (DCs). Our previous studies indicate that DCs pulsed with recombinant fusion proteins of antigenic fragment and HSP70-like protein 1 (HSP70L1) are potent in stimulating antigen-specific Th1 responses. We herein evaluated the cytotoxic T cell (CTL) response by an intracellular approach of priming DCs with transfection of recombinant adenovirus-expressing the fusion gene of the 576-699 fragment of carcinoembryonic antigen (CEA) and HSP70L1. As compared with DCs pulsed with extracellular fusion protein, the DCs transfected with recombinant adenovirus expressing the fusion gene displayed equivalent mature phenotypes but less inflammatory appearance. However, the transfected DCs were superior to the pulsed DCs in inducing CEA-specific CTLs. Consistently, immunization of HLA-A2.1/H-2Kb transgene mice with the transfected DCs could induce more quantities of HLA-A2.1-restricted CEA-specific CTLs, protecting nude mice more significantly from human CEA-expressing colon tumor challenge when adoptively transferred. Mechanistic investigation indicated that intracellular expression of the fusion protein empowered the transfected DCs by activation of STAT1 possibly via inducing IFN-β and ERK pathways. Therefore, the more potent ability to induce anti-CEA CTL responses enables the DCs, which transfected with recombinant adenovirus expressing the fusion gene of antigenic CEA fragment and Th1 adjuvant, as an alternative promising approach for the immunotherapy of CEA-positive tumors.