Introduction 125I seeds release a continuous low dose rate radiation (CLDR) used to treat patients with lung cancer;however, the effect is not as well characterized as other treatments, such as external beam radiation therapy (EBRT).Therefore, we compared the biological effects of CLDR to EBRT in vitro to determine its mechanism of action.Methods H520 lung cancer cells were exposed to equivalent doses of EBRT and CLDR.An X-ray linear accelerator was used to administer EBRT and125I seeds were used for CLDR.Cell survival, cell death, and changes to the cell cycle were monitored to determine the response to radiation.In addition, double-stranded DNA break damage and repair mechanisms were determined using real-time PCR and western blotting.Results The colony formation of H520 cells depreciated rapidly as radiation doses increased, especially for CLDR treatment (RBE (CLDR/EBRT) =1.66).Similar results were observed by MTS proliferation assay.After radiation at 4 Gy, the percentage of cells arrested in G2/M phase was also higher in CLDR treated cells.In addition, we found markedlyincreased apoptotic cells following CLDR by FCM with PI/Annexin Ⅴ staining..At the molecular level, p-H2AX (Ser139) was significantly elevated after CLDR.Neither CLDR nor EBRT affected DNA-PK, Ku80, or Ku70 mRNA expression levels;however, the protein expression of Ku70 and Ku80 was reduced following CLDR.Finally, the phosphorylation of DNA-PKcs was not affected by CLDR treatment.Conclusions CLDR with 125I seeds more efficiently inhibited H520 lung cancer cell growth than EBRT.CLDR caused severe DNA damage in H520 lung cancer cells by activating H2AX, limiting double-strand break repair by downregulating Ku70 and Ku80 protein expression, and inhibiting the function of DNA-PKcs.