In this study, we investigated the potential of combined treatment with temozolomide (TMZ) chemotherapy and tumor antigen-pulsed dendritic cells (DCs) and the underlying immunological factors of TMZ chemoimmunotherapy with an intracranial GL26 glioma animal model. The combined treatment enhanced the tumor-specific immune responses and prolonged the survival more effectively than either single therapy in GL26 tumor-bearing animals. Apoptosis was induced in the tumors of the animals by the treatment with TMZ. Calreticulin (CRT) surface exposure was detected by immunofluorescence staining of TMZ-treated GL26 cells. TMZ chemotherapy increased tumor antigen cross-priming from tumor cells, leading to cross-priming of tumor antigen-specific CD4(+) T cells and CD8(+) T cells. This chemotherapy appeared to suppress the frequency of CD4(+) CD25(+) regulatory T cells (Treg). Moreover, this combined therapy resulted in an increase in the tumor infiltration of CD4(+) and CD8(+) T cells. Collectively, the findings of this study provide evidence that the combination of TMZ chemotherapy and treatment with DC-based vaccines leads to the enhancement of antitumor immunity through increased tumor-specific immune responses via the cross-priming of apoptotic tumor cell death mediated by CRT exposure and, in part, the suppression of Treg. Therefore, CRT exposure, regulatory T cells, and cross-priming by TMZ chemotherapy may be immunological factors related to the enhancement of the antitumor effects of chemoimmunotherapy in an experimental brain tumor model.