Abstract Abstract Radiation therapy continues to play a central role in the management of brain tumors. The radioresistance of high-grade gliomas remains one of the reasons for the failure of treatment, which would otherwise require tumoricidal doses beyond the normal tissue tolerance and beyond safe clinical practice. Thus, determining the cellular, molecular and biochemical mechanisms underlying radiation resistance becomes critical for successful treatment of these tumors. For this purpose, glioma cell line U87 were treated with fractionated doses of radiation and the resistant clones isolated from the surviving tumor cells were characterized. The radioresistant U87 cells showed enhanced aggressiveness and clonogenic survival with an increased expression of HER2 and CD133, glioma cancer stem cell surface markers, as well as elevated mitochondrial membrane potential but lower oxygen consumption and ATP generation. In addition, compared with the wild type U87 cells, the phosphorylation of mitochondrial Stat3 at Serine 727 was decreased in the radioresistant U87 cells, indicating a role for mitochondrial Stat3 in the radioresistance mechanisms. Additionally, mutation of Serine 727 of Stat3 into Alanine resulted in increased radiosensitivity. These results demonstrate that the radioresistant glioma cells surviving routine radiotherapy are enriched with cancer stem cells expressing CD133 and HER2. The radioresistant phenotype is linked with a lowered mitochondrial metabolism and increased anti-apoptotic function due to inhibition of mitochondrial Stat3 phosphorylation. Thus, improving the mitochondrial metabolism by enhancing mitochondrial Stat3 phosphorylation may serve as an effective approach to re-sensitize the radioresistant glioma tumors. Citation Format: Ming Fan, Yan Shi, Jian Jian Li. Mitochondrial metabolism and phosphorylation of Stat3 in radioresistant cancer stem cells isolated from glioma U87 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 444. doi:10.1158/1538-7445.AM2013-444