Abstract: Objective To determine the effect of downregulation of 53BP1 expression on cell growth and radiosensitivity in laryngeal carcinoma Hep-2 cells. Methods HEP-2 cell lines were established with a stable knockdown of 53BP1 with short hairpin RNA (shRNA). The level of expressed protein of negative control group（NC），wild type Hep-2 and downregulation of 53BP1 （P6）group was determined by Western blotting. Proliferation on normal conditions was detected by MTT. Radiosensitivity and growth of cells were detected by flow cytometry. Results A stable 53BP1 downregulated cell line P6 was established. Similar cell growth was observed between the 53BP1 downregulated cells and the control cells. The downregulation of 53BP1 reduced the number of clonogenic cells exposed to radiation. Compared with wild type Hep-2 group and NC group,Western blot results showed a decrease in 53BP1 protein level in P6 group (P<0.05)，reducing the ratio of arrest of G2/M with radiation dose increased (P<0.05). MTT results revealed the lower 53BP1 protein level did not affect the cell proliferation. After exposure to 0, 2, 6, 10 Gy ionizing radiation (IR), P6 cells had lower, radiosensitivity parameters (D0, SF2, Dq, N) than controls and wild type Hep-2 (P<0.05). Conclusion RNA interference could effectively down-regulate the expression of 53BP1 and reduce arrest of G2/M phase, thus enhancing the radiosensitivity of Hep-2 cell line.