Abstract: Objective To determine the function of human papillomavirus (HPV) E7 in DNA damage response of cervical intraepithelial neoplasia (CIN) Ⅲ cells. Methods Samples of CIN Ⅲ and child foreskin tissues were collected, with pathologically confirmed HPV positive and negative, respectively. Collagenase A was used for digesting tissues prior to primary culture. The HPV negative cells were infected with lentivirus E7 and pLV. Proteins (53BP1, NBS1, BRCA1 and RPA32) responsive to DNA double break damages were detected by indirect immunofluorescent staining after 0-8 h treatment with X-ray (2 or 5 Gy). Results After treatment with 2 or 5 Gy X-ray, 53BP1, NBS1, BRCA1 and RPA32 foci in HPV⁺ cells increased compared with HPV^- cells (P<0.05). Less 53BP1, RPA32, BRCA1 and NBS1 foci positive cells (foci>5) were found in E7 infected cells than in pLV infected cells (P<0.05). Both of them reached the peak at 6 h (2 Gy) or 4 h (5 Gy). Conclusion We have successfully established a model to detect the function of HPV E7 in DNA damage response using primary culture of CIN fibroblasts. With the progression of CIN, HPV E7 can inhibit DNA double break repair.