Mitochondrial DNA (mtDNA) repair systems are thought to be associated with the susceptibility of cancer cells to anticancer agents. The present study investigated the relationship between the susceptibility to gamma-rays and the mtDNA repair ability of oral squamous cell carcinoma (OSC) cell lines. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and mtDNA common deletion in both nuclear and mitochondrial DNA of OSC-2, OSC-3 and OSC-6 cells (radio-sensitive cell lines) after gamma-ray-irradiation were higher than those of OSC-1, OSC-4 and OSC-5 cells (radio-resistant cell lines). Compared with OSC-2, OSC-3 and OSC-6 cells, OSC-1, OSC-4 and OSC-5 cells had higher levels of activity of phosphoinositide-3 kinase (PI-3K)/Akt and more strongly expressed 8-hydroxyguanine DNA glycosylase (OGG1), DNA polymerase gamma (POLG) and mitochondrial transcription factor A (Tfam). Down-regulation of these mtDNA-repair-associated molecules by the RNA interference technique enhanced the susceptibility of OSC-2 and OSC-5 cells to gamma-rays, and the expression of Tfam and POLG was down-regulated by inhibitors of PI-3K/Akt signaling. These results indicate that the inhibition of mtDNA repair capacity by PI-3K/Akt signal inhibitors and OGG1 down-regulator in cancer cells may be a useful strategy for cancer treatment when combined with ionizing irradiation and chemotherapeutic drugs.