Glioblastoma multiforme (GBM) is the most malignant intracranial tumor in adults and its unique pathology leads to limited therapeutic benefits.1,2 Mitochondrial fusion and fission play an important role in carcinogenesis; fragmented mitochondria promote tumor cell proliferation and prolonged mitochondria lead to tumor cell apoptosis.3 Therefore, profiling the function and prognostic value of mitochondrial dynamics-related genes (MDRGs) is of great interest for GBM precision treatment. Here we focused on the expression, function, and genetic alterations of MDRGs and identified new DNA methylation sites being significantly associated with the survival of GBM patients using available data in public databases. We then constructed the tumor prognostic model that accurately forecast the survival of GBM patients based on MDRGs' signature. Furthermore, it was demonstrated that the expression of MDRGs and risk factors served as independent indexes to estimate the level of immune infiltration in tumor microenvironment and response to targeted immune checkpoints in GBM patients. Notably, we filtered out acetaminophen targeting risk genes as a candidate drug for GBM treatment after clarifying risk genes' contribution to the cancer process at the single-cell level. Overall, the new biomarkers, prognostic model, and targeted drugs characterized in this study provide a novel perspective for GBM management.