Anti-tumour therapies eliminate proliferating tumour cells by induction of DNA damage, but genomic aberrations or transcriptional deregulation may limit responses to therapy. Glioblastoma (GBM) is a malignant brain tumour, which recurs inevitably due to chemo- and radio-resistance. Human RecQ helicases participate in DNA repair, responses to DNA damage and replication stress. We explored if a helicase RECQL4 contributes to gliomagenesis and responses to chemotherapy. We found upregulated RECQL4 expression in GBMs associated with poor survival of GBM patients. Increased levels of nuclear and cytosolic RECQL4 proteins were detected in GBMs on tissue arrays and in six glioma cell lines. RECQL4 was detected both in cytoplasm and mitochondria by Western blotting and immunofluorescence. RECQL4 depletion in glioma cells with siRNAs and CRISPR/Cas9 did not affect basal cell viability, slightly impaired DNA replication, but induced profound transcriptomic changes and increased chemosensitivity of glioma cells. Sphere cultures originated from RECQL4-depleted cells had reduced sphere forming capacity, stronger responded to temozolomide upregulating cell cycle inhibitors and pro-apoptotic proteins. RECQL4 deficiency affected mitochondrial network and reduced mitochondrial membrane polarization in LN18 glioblastoma cells. We demonstrate that targeting RECQL4 overexpressed in glioblastoma could be a new strategy to sensitize glioma cells to chemotherapeutics.