Development of drug resistance after standard chemotherapy for glioblastoma multiforme (GBM) with temozolomide (TMZ) is associated with poor prognosis of GBM patients and is at least partially mediated by a direct DNA repair pathway involving O6-methylguanine methyltransferase (MGMT). This enzyme is under post-translational control by a multisubunit proteolytic cellular machinery, the 26S proteasome. Inhibition of the proteasome by bortezomib (BZ), a boronic acid dipeptide already in clinical use for the treatment of myeloma, has been demonstrated to induce growth arrest and apoptosis in GBM cells. In this study we investigated the effect of sequential treatment with BZ and TMZ on cell proliferation-viability and apoptosis of the human T98G and U87 GBM cell lines. We also tested for an effect of treatment on MGMT expression and important upstream regulators of the latter, including nuclear factor kappa B (NFκB), p44/42 mitogen-activated protein kinase (MAPK), p53, signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor 1α (HIF-1α). The sequence of drug administration for maximal cytotoxicity favored BZ prior to TMZ in T98G cells while the opposite was the case for U87 cells. Maximal efficacy was associated with downregulation of MGMT, reduced IκBα-mediated proteasome-dependent nuclear accumulation of NFκB, attenuation of p44/42 MAPK, AKT and STAT3 activation, and stabilization of p53 and inactive HIF-1α. Collectively, these results suggest that proteasome inhibition by BZ overcomes MGMT-mediated GBM chemoresistance, with scheduling of administration being critical for obtaining the maximal tumoricidal effect of combination with TMZ.