DNA-dependent protein kinase (DNA-PK), including Ku80, Ku70 and DNA-PK catalytic subunit (DNA-PKcs), is the key protein in non-homologous end-joining (NHEJ) after DNA double-strand breaks (DSBs) appear. In this study, small hairpin interfering RNAs (siRNAs) targeting Ku80 and DNA-PKcs were used both individually and in combination, to explore the effects of these DSB proteins on He La cell functional changes after X-ray irradiation. He La cells co-transfected with Ku80-siRNA and DNA-PKcs-siRNA were more radiosensitive than the ones transfected individually. He La in the absence of Ku80 and pretreated with LY294002, a chemically specific PI 3-kinase inhibitor, resulted in cells that were even more sensitive to X-rays than HeLa/Ku80-siRNA transfected with DNA-PKcs-siRNA. The cells inhibited by Ku80 either individually or in combination with DNA-PKcs showed cell accumulation in the G2/M phase 48 h post-irradiation, similarly to control cells. However, cells transfected with DNA-PKcs-siRNA or pretreated with LY294002 had a prolonged G2/M delay, suggesting the accumulation of significant un-repaired DNA damage following inhibition of DSB repair proteins. In conclusion, these data indicate that the role of Ku80 in DSB repair could be compensated by other DSB repair proteins: co-inhibition would be a suitable strategy to enhance the radiosensitivity of cancer cells.