The Ras activation contributes to radioresistance, but the mechanism is unclear. This article shows that the expression of the dominant-positive H-Ras increased the Ku80 level, which is one of the key enzymes involved in repairing dsDNA breaks (DSB). After exposing the cells to ionizing radiation and analyzing them using an electrophoretic mobility shift assay and pulsed-field gel electrophoresis, it was found that activated H-Ras expression in NIH3T3 cells increases the DNA-binding activity of Ku80 and increases the DSB repair activity. Ku80 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DSBs and suppress the oncogenic H-Ras-mediated resistance of the cells to gamma-ray irradiation, whereas Ku80 overexpression in the NIH3T3 cells significantly increased the radioresistance. These results suggest that the Ku80 expression induced by oncogenic H-Ras seems to play an important role in protecting cells against gamma-ray irradiation.