Telomere maintenance enables the indefinite proliferation potential of cancer cells and is among the most frequent alterations in human glioblastoma (GBM). Here, Bejarano et al. report that expression of the telomere binding protein TRF1 — essential for telomere capping and protection — is upregulated in mouse and human GBM compared with normal brain tissue. In mouse GBM models, Trf1 deletion increased telomeric damage, reduced stemness and inhibited GBM initiation and progression. Small-molecule TRF1 inhibitors mimicked these anticancer effects in patient-derived human xenografts.