Objective:To study the radioresistance of esophageal cancer stem cells. Methods:The ECA109 cells were cultured in serum-free medium to enrich esophageal stem-like cells. MTT assay was performed to detect the multiplication capacity of cancer cells treated with different concentrations of cyclooxygenase-2 (COX-2) selective inhibitor NS398 (2.5,5.0,10.0,20.0,40.0 and 80.0 μmol/L). Sensitization effects of parental cells and spheroid cells were evaluated by clone formation assay. Sphere-forming assay was aimed to analyze the effect of NS398 combined with X-ray radiation on cell spheres formation. Western blot was performed to determine the expression level of β-catenin. Results:NS398 inhibited the proliferations of parental cells and spheroid cells in a time-and concentration-dependent way. After treated with 20 μmol/LNS398,Do,Dq and SF2 values of parental cells were all decreased (P < 0.05),and the sensitization enhancement ratio (SER) was 1.17. Do and SF2 values of spheroid cells were decreased (P < 0.05),and the SER was 1.12. Formation rate of the cell spheres was increased after irradiation (P < 0.05). Compared with the radiation alone group,the formation rate of the cell spheres and β-catenin expression of parental cells and spheroid cells were decreased in the combination group (t = 7.01,P < 0.01;t = 10.15,P < 0.01;t = 3.225,P < 0.05). Conclusion:The sensitization effect of spheroid cells is less than parental cells,which indicates that spheroid cells has the radioresistance. It may be correlated with the inhibition of the expression level of β-catenin.