We studied the feasibility, efficacy, and mechanisms of dendritic cell (DC) immunotherapy against murine malignant glioma in the experimental GL261 intracranial (IC) tumor model. When administered prophylactically, mature DCs (DCm) ex vivo loaded with GL261 RNA (DCm-GL261-RNA) protected half of the vaccinated mice against IC glioma, whereas treatment with mock-loaded DCm or DCm loaded with irrelevant antigens did not result in tumor protection. In DCm-GL261-RNA-vaccinated mice, a tumor-specific cellular immune response was observed ex vivo in the spleen and tumordraining lymph node cells. Specificity was also shown in vivo on the level of tumor challenge. Depletion of CD8(+) T-cells by anti-CD8 treatment at the time of tumor challenge demonstrated their essential role in vaccine-mediated antitumor immunity. Depletion of CD25(+) regulatory T-cells (Tregs) by anti-CD25 (aCD25) treatment strongly enhanced the efficacy of DC vaccination and was itself also protective, independently of DC vaccination. However, DC vaccination was essential to protect the animals from IC tumor rechallenge. No long-term protection was observed in animals that initially received aCD25 treatment only. In mice that received DC and/or aCD25 treatment, we retrieved tumor-specific brain-infiltrating cytotoxic T-lymphocytes. These data clearly demonstrate the effectiveness of DC vaccination for the induction of long-lasting immunological protection against IC glioma. They also show the beneficial effect of Treg depletion in this kind of glioma immunotherapy, even combined with DC vaccination.