Glioblastoma presents a formidable obstacle for developing immunotherapeutic strategies due to a highly immunosuppressive systemic and micro-environment. Antibody blockade of co-inhibitory receptors CTLA-4 and PD-1 has been shown to overcome immunosuppression in clinical trials of various solid tumors. We evaluated the anti-tumor efficacy of murine antibodies targeting CTLA-4 and/or PD-1 in the GL261 orthotopic, immunocompetent mouse model of glioblastoma. We observed tumor regression and long-term survival in 15% and 50% of mice treated with anti-CTLA-4 or anti-PD-1 as single agents, respectively. Combination therapy resulted in 75% of animals surviving long-term and tumor-free. Importantly, similar efficacy was observed with both early (day 7) or late (day 14) onset of treatment, thus this therapeutic strategy was effective on established tumors. Furthermore, resistance to tumor re-challenge demonstrated the generation of immunological memory in treated mice. Upon investigating the systemic and tumor immunological profile, we discovered that combination therapy increased CD8+ and NK effector cells and decreased suppressive immune cells at the tumor site and draining lymph node. In addition, combination therapy increased the serum levels of various monocyte chemo-attractant chemokines involved in immune activation. Our data provide strong support for the evaluation of immune checkpoint blockade in glioblastoma patients, and predicts potential prognostic serum biomarkers.