Abstract Glioblastoma (GBM) cancer stem cells (CSC) are primarily responsible for metastatic dissemination, resistance to therapy, and relapse of GBM, the most common and aggressive brain tumor. Development and maintenance of CSCs require orchestrated metabolic rewiring and metabolic adaptation to a changing microenvironment. Here, we show that cooperative interplay between the mitochondrial chaperone TRAP1 and the major mitochondria deacetylase sirtuin-3 (SIRT3) in glioma stem cells (GSC) increases mitochondrial respiratory capacity and reduces production of reactive oxygen species. This metabolic regulation endowed GSCs with metabolic plasticity, facilitated adaptation to stress (particularly reduced nutrient supply), and maintained “stemness.” Inactivation of TRAP1 or SIRT3 compromised their interdependent regulatory mechanisms, leading to metabolic alterations, loss of stemness, and suppression of tumor formation by GSC in vivo. Thus, targeting the metabolic mechanisms regulating interplay between TRAP1 and SIRT3 may provide a novel therapeutic option for intractable patients with GBM. Significance: Discovery and functional analysis of a TRAP1–SIRT3 complex in glioma stem cells identify potential target proteins for glioblastoma treatment.