Glioblastoma multiforme (GBM) almost invariably acquires an invasive phenotype, resulting in limited therapeutic options. Protein palmitoylation markedly affects tumorigenesis and malignant progression in GBM. The role of protein palmitoylation in GBM, however, has not been systematically reported. This study aimed to investigate the effect of protein palmitoylation on GBM cell survival and the cell cycle. In this study, most palmitoyltransferases were upregulated in GBM and its cell lines, and protein palmitoylation participated in signaling pathways controlling cell survival and the GBM cell cycle. Inhibition of protein palmitoylation with substrate-analog inhibitors, that is, 2-bromopalmitate, cerulenin, and tunicamycin, induced G₂ cell cycle arrest and cell death in GBM cells through enhanced endoplasmic reticulum (ER) stress. These effects are primarily attributed to the palmitoylation inhibitors activating pro-apoptotic pathways and ER stress signals. Further analysis revealed was the accumulation of SUMOylated XBP1 (X-box binding protein 1) and its transcriptional repression, along with a reduction in XBP1 palmitoylation. Taken together, the present results indicate that protein palmitoylation plays an important role in the survival of GBM cells, further providing a potential therapeutic strategy for GBM.