Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD-or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM. Human glioblastoma multiforme (GBM) is the most lethal primary brain cancer in adults. In the United States alone, approximately 12,000 patients are diagnosed with GBM each year 1. The standard care for GBM includes surgical resection followed by concurrent external beam radiotherapy (with a total dose of 60 Gy) together with temozolomide (TMZ), as a supplemental DNA-methylating agent. It is followed by one year of adjuvant treatment with TMZ 2. While external beam radiation therapy efficiently targets brain tumors, it could be accompanied by severe side effects, such as encephalopathy, strong cognitive and memory deficits 3-8. Despite advances in GBM radiotherapy, outcomes remain poor with a median survival rate of 12-15 months after initial diagnosis 9,10. In the past 10 years, survival in the treatment of this disease did not increase significantly. In spite of intense investigation on the gene drivers of GBM tumorigenesis, targeted therapies against these drivers have remained inefficient 11. Numerous studies in the recent years revealed pronounced cytotoxic effects of cannabinoids [cannabidiol (CBD) and Δ 9-tetrahydrocannabinol (THC)] for GBM in cell culture and experimental animals 12-16. Furthermore, preclinical studies demonstrated that treatment of GBM by combinations of cannabinoids and γ-irradiation or cannabinoids with TMZ was more effective against cancer cells than non-transformed cells in the brain 17-19. To further increase efficiency, specificity, and safety of the treatment, we address in the present study the question whether autophagy could play a protective role in the survival of CBD/γ-irradiation...