We investigated the relationship between significantly different genes of the mitochondria-to-nucleus retrograde signaling pathway (RTG) in H1299 ρ⁰ cells (mtDNA depleted cell) and compared their radiosensitivity to that of parental ρ cells, to determine the possible intervention targets of radiosensitization. ρ⁰ cells were depleted of mitochondrial DNA by chronic culturing in ethidium bromide at low concentration. Radiosensitivity was analyzed using clonogenic assay. Western blot was used to analyze the cell cycle-related proteins, serine/threonine kinase ataxia telangiectasia mutant (ATM), ataxia telangiectasia and Rad3-related protein (ATR) and cyclin B1 (CCNB1). The γ-H2AX foci were detected using confocal fluorescence microscopy. RNA samples were hybridized using the Agilent human genome expression microarray. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for Gene Ontology (GO) Consortium and pathway annotations of differentially expressed genes, respectively. The H1299 ρ⁰ cells were found to be more radioresistant than ρ cells. The ATP production of H1299 ρ⁰ cells was lower than that of the ρ cells before or after irradiation. Both H1299 ρ⁰ and ρ cells had higher ROS levels after irradiation, however, the radiation-induced ROS production in ρ⁰ cells was significantly lower than in ρ cells. In addition, the percentage of apoptosis in H1299 ρ⁰ cells was lower than in ρ cells after 6 Gy irradiation. As for the cell cycle and DNA damage response-related proteins ATM, ATR and CCNB1, the expression levels in ρ⁰ cells were significantly higher than in ρ cells, and there were less γ-H2AX foci in the ρ⁰ than ρ cells after irradiation. Furthermore, the results of the human genome expression microarray demonstrated that the phosphorylated protein levels of the NF-κB/PI3K/AKT2/mTOR signaling pathway were increased after 6 Gy irradiation and were decreased after treatment with the AKT2-specific inhibitor MK-2206 combined with radiation in H1299 ρ⁰ cells. MK-2206 treatment also led to an increase in pro-apoptotic proteins. In conclusion, these results demonstrate that mtDNA depletion might activate the mitochondria-to-nucleus retrograde signaling pathway of NF-κB/PI3K/AKT2/mTOR and induce radioresistance in H1299 ρ⁰ cells by evoking mitochondrial dysfunctions.