AbstractBackgroundRadiotherapy is one of the main strategies for the treatment of esophageal squamous cell carcinoma (ESCC). However, treatment failure often occurs due to the emergence of radioresistance. In this study, we report a key regulator of radiation sensitivity, termed TAB182 that may become an ideal biomarker and therapeutic target to overcome radioresistance.Materials and MethodsBy applying qRT‐PCR and immunohistochemical staining, the expression of TAB182 was detected in patient tissues. We next assessed the influence of TAB182 downregulation to radiosensitivity using clonogenic survival assay and γ‐H2A.X foci analysis in TE‐1, TE‐10, and radioresistant TE‐1R cell lines after ionizing radiation. To unveil the mechanism underlying, TAB182 interacting proteins were identified by mass spectrometry following co‐immunoprecipitation. Furthermore, flow cytometry and western blot assay were applied to validate the identified proteins.ResultsOur results demonstrated that the expression of TAB182 is higher in cancer tissues than normal tissues and elevated expression of TAB182 correlates with poor outcomes of postoperative radiotherapy. Downregulation of TAB182 sensitized cancer cells to ionizing radiation, particularly in radioresistant TE‐1R cells that spontaneously overexpress TAB182. Mechanically, TAB182 interacts with FHL2 to induce G2‐M arrest through wiring the CHK2/CDC25C/CDC2 signaling pathway. Finally, overexpression of shRNA‐resistant TAB182 restored the checkpoint and radioresistance.ConclusionTAB182 potentiates the radioresistance of ESCC cells by modulating the G2‐M checkpoint through its interaction with FHL2. Thus, TAB182 may become an ideal biomarker and therapeutic target of ESCC radiotherapy.