Background: Genetic studies on mice have demonstrated that the key regulator of DNA damage in mammalian cells is the histone H2A variant, H2AX. We hypothesize that knockdown of H2AX will cause DNA damage pathway defects and may be able to increase the sensitivity to radiotherapy. Methods: The formation of foci and the interaction of several important proteins in esophageal cancer ECA109, triggered by irradiation, were detected by immunofluorescence staining and Co-immunoprecipitation (Co-IP) assay before and after H2AX silencing. Clone formation assay was employed to detect cell radiosensitivity and cloning formation ability also before and after H2AX silencing. Cell cycle distribution and apoptosis were detected by flow cytometry. We constructed a nude mice esophageal cancer model and detected the above contents in vivo. Results: H2AX and several proteins could form foci in nuclear triggered by irradiation and establish a relationship in vitro. The foci reduced after H2AX silencing. H2AX silencing could lead to radiosensitization via a colony-forming test. The apoptosis rate increased and the cell cycle was blocked in G2-M stage after H2AX silencing in vivo. The tumor volume was decreased in the H2AX silenced group after irradiation, while the tumor only slowed down the growth rate in the control groups. Conclusions: Knockdown of H2AX induced radiosensitization of esophageal cancer ECA109 cells both in vitro and in vivo. The mechanisms include defective cell cycle checkpoints and abolishment of foci formation for several important mediator and effector proteins in the DNA damage response to irradiation (IR).