Glioblastoma (GBM) currently has a dismal prognosis. GBM cells that survive radiotherapy contribute to tumor progression and recurrence with metabolic advantages. Here, we show that diacylglycerol kinase B (DGKB), a regulator of the intracellular concentration of diacylglycerol (DAG), is significantly downregulated in radioresistant GBM cells. The downregulation of DGKB increases DAG accumulation and decreases fatty acid oxidation, contributing to radioresistance by reducing mitochondrial lipotoxicity. Diacylglycerol acyltransferase 1 (DGAT1), which catalyzes the formation of triglycerides from DAG, is increased after ionizing radiation. Genetic inhibition of DGAT1 using short hairpin RNA (shRNA) or microRNA-3918 (miR-3918) mimic suppresses radioresistance. We discover that cladribine, a clinical drug, activates DGKB, inhibits DGAT1, and sensitizes GBM cells to radiotherapy in vitro and in vivo. Together, our study demonstrates that DGKB downregulation and DGAT1 upregulation confer radioresistance by reducing mitochondrial lipotoxicity and suggests DGKB and DGAT1 as therapeutic targets to overcome GBM radioresistance.