Background: Benefit from temozolomide chemotherapy in glioblastoma is essentially limited to patients with tumors with O⁶-methylguanine DNA methyltransferase (MGMT) promoter methylation. The impact of the MGMT status on chemosensitivity may be modulated by telomerase reverse transcriptase (TERT) promoter mutations which affect two regions of the TERT promoter, designated as C228T and C250T. Methods: TERT promoter mutation status and TERT activity were determined in a panel of glioma cell lines and correlated with sensitivity to irradiation or temozolomide. TERT status alterations were induced using sh-mediated gene TERT silencing or wildtype TERT overexpression. TERT mutation and MGMT promoter methylation status were also determined in a clinical patient cohort from the German Glioma Network. Results: C228T-mutant glioma cell lines (n = 8) express higher levels of TERT mRNA (mean 0.046±0.012 vs 0.012±0.004 arbitrary units, p = 0.049) and exhibit higher TERT catalytic activity (mean 122±16 vs 53±11 arbitrary units, p = 0.022) than C250T-mutant glioma cell lines (n = 5). C228T-mutant glioma cell lines are also more sensitive to irradiation (mean ED90 4.6±0.7 versus 7.1±0.8 Gy, p = 0.039) or temozolomide (mean EC50 101.6±58.5 versus 295.2±53.8 µM, p = 0.045) in vitro. Targeted alterations of TERT status result in profound changes in radiosensitivity and chemosensitivity: TERT gene silencing is protective whereas TERT overexpression sensitizes to either genotoxic treatment. Consistent with these preclinical observations, patients with C228T TERT mutation and MGMT promoter methylation appeared to derive more benefit from temozolomide chemoradiotherapy (median overall survival 26.5 months, 95% CI 20.3-32.7) than patients with the C250T mutation (median overall survival 16.2 months, 95% CI 8.5-23.8) or patients without TERT mutation (median overall survival 23.7 months, 95% CI 18.7-28.8). Conclusions: These data refine current models on how TERT and MGMT interact to determine outcome in human glioblastoma and illustrate how future targeted interventions focusing on TERT and MGMT may help to tailor pharmacotherapy of glioblastoma.