Proteasome inhibitors are emerging as a new class of cancer therapeutics, and bortezomib has shown promise in the treatment of multiple myeloma and mantle cell lymphoma. However, bortezomib has failed to have an effect in preclinical models of glioma. NPI-0052 is a new generation of proteasome inhibitors with increased potency and strong inhibition of all three catalytic activities of the 26S proteasome. In this article, we test the antitumor efficacy of NPI-0052 against glioma, as a single agent and in combination with temozolomide and radiation using five different glioma lines. The intrinsic radiation sensitivities differed for all the lines and correlated with their PTEN expression status. In vitro, NPI-0052 showed a dose-dependent toxicity, and its combination with temozolomide resulted in radiosensitization of only the cell lines with a mutated p53. The effect of NPI-0052 as a single agent on glioma xenografts in vivo was only modest in controlling tumor growth, and it failed to radiosensitize the glioma xenografts to fractionated radiation. We conclude that NPI-0052 is not a suitable drug for the treatment of malignant gliomas despite its efficacy in other cancer types.