Topoisomerase II alpha (Top2alpha) is an attractive candidate to be used as a tumor antigen for cancer immunotherapy, because it is abundantly expressed in various tumors and serves as a target for a number of chemotherapeutic agents. In this study, we demonstrated the immunogenicity of Top2alpha, using dendritic cells (DC) electroporated with RNA encoding the Top2alpha C-terminus (Top2alphaCRNA/DC). Top2alphaCRNA/DC were able to demonstrate in vitro stimulation of T cells from mice that were previously vaccinated with Top2alpha-expressing tumor lysate-pulsed DC. Vaccination with Top2alphaCRNA/DC induced Top2alpha-specific T cell responses in vivo as well as antitumor effects in various murine tumor models including MC-38, B16F10, and GL26. DC pulsed with p1327 (DSDEDFSGL), defined as an epitope presented by H-2K(b), also induced Top2alpha-specific immune responses and antitumor effects. Based on these data, Top2alpha is suggested to be a universal target for cancer immunotherapy.