Abstract Background Dendritic Cells (DC) are potent antigen presenting cells with the ability to prime naïve T cells and convert them to cytotoxic T-lymphocytes (CTL). We evaluated the capability of autologous DCs transfected with total tumor and normal RNA to induce cytotoxic CTL as the preliminary step to design a DC-based vaccine in the esophageal squamous cell carcinoma (ESCC). Methods Monocytes-derived DCs were electroporated with either total tumor RNA or normal RNA. T cells were then primed with tumor RNA transfected DCs and lytic effects of the generated CTL were measured with Cytotoxicity assay and IFN-γ Release Elispot assay. Results Cytotoxicity was induced against DCs loaded with tumoral RNA (%24.8 ± 5.2 SEM) while in normal RNA-loaded DCs, it was minimal (%6.1 ± 2.4 SEM) and significantly lower (p < 0.05). INF-γ secretion was more than 2-folds higher in tumoral RNA-loaded DCs when compared with normal RNA-loaded DCs (p < 0.05). Conclusion Electroporating DCs with tumor RNA generated tumor antigen presenting cells which in turn enhanced cytotoxic effects of the T cells against ESCC. This may be a useful autologous ex vivo screening tool for confirming the lytic effects of primed T cells on tumors and evaluate probable further adverse effects on noncancerous tissues. These data provide crucial preliminary information to establish a total tumor RNA-pulsed DC vaccine therapy of ESCC.