Abstract Glycogen storage has been demonstrated in multiple cancer cell lines and is upregulated in response to hypoxia both in vitro and in vivo. Evidence supports a potential role of the “glycogen shunt”, that describes the cycling of glucose through glycogen to subsequently fuel glycolysis, in brain tumors. Glioblastoma (GBM) is the most common and most deadly central nervous system tumor, and is characterized by hypoxia and metabolic reprogramming. We found that inhibition of glycogen degradation by downregulation of glycogen phosphorylase liver isoform (PYGL) in GBM cells led to a decrease in clonogenic growth and sensitization to ionizing radiation (IR) doses of 10-12 Gy. Control cells also showed growth impairment but resumed growth 10 days after IR exposure, while PYGL knockdown cells did not. To understand these different responses to high dose IR, we studied the intra-cellular effects of IR in control and PYGL knockdown GBM cells. Five days after IR exposure of PYGL knockdown cells, mitotic catastrophe and a senescence-like morphology were seen. Dysregulation of autophagy, accumulation of elongated and fragmented mitochondria, decrease in oxygen consumption rate and extracellular acidification rate, and increased pAMPK and pACC expression were shown in irradiated PYGL knockdown cells compared to control cells, suggesting metabolic stress. In human GBMs, PYGL mRNA expression was higher than in normal brain tissue and low PYGL mRNA expression was correlated with improved survival rates (hazard ratio 1.32, 95% confidence interval 1.08 - 1.61, P = 0.006). Immunohistochemical PYGL expression was also increased in human GBM versus normal brain tissue. In conclusion, inhibition of glycogen degradation sensitizes GBM cells to high dose IR, the cornerstone of GBM treatment, and may provide a way forward to improve treatment of primary GBMs. Citation Format: Anne M. Hendriks, Christos E. Zois, Syed B. Haider, Elisabete Pires, Dimitra Kalamida, Christoffer Lagerholm, John Morris, James S. McCullagh, Rudolf S. Fehrmann, Wilfred F. den Dunnen, Adrian L. Harris, Mathilde Jalving. Inhibition of glycogen degradation sensitizes glioblastoma cells to ionizing radiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 240.