Objective:To clarify the role of classic Wnt signaling pathway in the radioresistance of esophageal cancer cells (ECC), and investigate the underlying mechanism, aiming to identify critical molecular targets for clinically enhancing the radiosensitivity of esophageal cancer.Methods:The radiosensitivity of four types of ECCs (EC9706, ECA109, KYSE70 and KYSE150) were assessed by colony formation assay. Western blot and RT-PCR were used to detect the activation of classical Wnt signaling pathway after irradiation. Classic Wnt signaling pathway activator (AZD2858) and inhibitor (XAV-939) were utilized to comprehensively evaluate the effect of classic Wnt signaling pathway on the radiosensitivity of ECCs. Cellular immunofluorescence staining was performed to detect the production and repair of DNA double-strand breaks (DSB), as well as the foci formation of DSB repair proteins after irradiation.Results:The results of colony formation assay showed that the radiosensitivity of four types of ECCs from high to low was EC9706, ECA109, KYSE70 and KYSE150. In KYSE150, a radioresistant cell type, the level of nuclear β-catenin and the transcription of c-Myc gene were significantly increased after irradiation (both P<0.05). However, in EC9706, a radiosensitive cell type, the level of nuclear β-catenin and c-Myc gene transcription were not affected by irradiation (both P>0.05). Moreover, EC9706 cells showed enhanced radioresistance in the presence of AZD2858( P<0.05), whereas XAV-939 treatment decreased the radioresistance in KYSE150 cells ( P<0.05). AZD2858 accelerated the DSB repair in EC9706 cells ( P<0.05), whereas XAV-939 delayed the DSB repair in KYSE150 cells ( P<0.05). Furthermore, the results of immunofluorescence staining showed that XAV-939 reduced the DSB repair capacity by inhibiting homologous recombination repair-related proteins (BRCA1 and RAD51) rather than non-homologous end junction repair-related proteins (Ku80 and XRCC4). Conclusions:The classic Wnt signaling pathway participates in the regulation of radiosensitivity in ECCs by regulating the homologous recombination repair of DSB after irradiation. Inhibition of the classic Wnt signaling pathway can counteract the radioresistance of ECCs and enhance the killing effect of irradiation on ECCs.