Radiation is a current standard treatment of glioma. The fractionated radiotherapy with low dose of radiation over weeks has been employed in glioma patients, while radiotherapy can only offer palliation due to the radioresistance. We cumulatively radiated a glioblastoma cell line, U87MG, and screened radioresistant glioma cells. A transcriptome sequencing was performed to analyze the transcription differences between the raidoresistant and control cells, which showed the mitochondria NADH–ubiquinone oxidoreductase (Complex I) subunits were up-regulated in the radioresistant cells. The copy numbers of mitochondria were increased in the radioresistant glioma cells. After using mitochondria Complex I inhibitors, rotenone and metformin, to treat glioma cells, we found the resistant glioma cells re-sensitized to radiation. These results demonstrate that Complex I is associated with the fractioned radiation-induced radioresistance of glioma and would be a potent target for clinical radiotherapy of glioma.