Abstract: Objective: To construct a stable transfected esophageal squamous cell carcinoma EC109 cell line overexpressing human ubiquitin cross-linking enzyme UbcH5a gene and investigate the mechanism of UbcH5a affecting the radiosensitivity of esophageal carcinoma.Methods: EC109 cells were divided into blank control group (no treatment), negative control group (transfected with pEGFP-C1 plasmid) and overexpression group(transfected with pEGFP-UbcH5a plasmid).Real-time fluorescence quantitative PCR (RT-qPCR) and western blotting were respectively used to detect the expression of UbcH5a mRNA and protein to verify the transfection efficiency.Clonogenic assay was used to detect the cell surviving fraction after 2 Gy X-ray irradiation(SF2), and immunofluorescence was used to detect DNA damage.The expression of DNA damage repair related proteins(ATM, ATR, p-ATM, p-ATR, Chk1, Chk2 and BRCA1) was detected by western blotting.Results: Compared with the blank control group, the expression levels of UbcH5a mRNA and protein were increased, SF2 was decreased, and DNA damage foci were increased in the overexpression group(all P< 0.05), while there was no significant change in the negative control group (P> 0.05).Compared with the negative control group, the expression of ATM, ATR, p-ATM, P-ATR, Chk1, Chk2 and BRCA1 in the overexpression group was significantly downregulated before and after X-ray irradiation (all P< 0.05).Conclusion: UbcH5a enhances the radiosensitivity of esophageal cancer cells by inhibiting the expression of DNA damage repair related proteins.