Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.