Radiotherapy (RT) is a traditional and important treatment for carcinoma of the esophagus along with surgery and chemotherapy. High mobility group box 1 (HMGB1) plays a crucial part in inhibiting the apoptosis of cancer cells after irradiation treatment. The present study, was designed to analyze the function of HMGB1 in esophageal cancer progression and elucidate the effects of HMGB1 on the radiosensitivity of human esophageal cancer cell lines. In the present study, an immunohistochemical evaluation of HMGB1 was performed on 77 biopsies, and the results revealed that HMGB1 overexpression was positively correlated with gross tumor volume (GTV), tumor-node-metastasis (TNM) stage, T classification, distant metastasis, and relapse and negatively correlated with patient survival rates, suggesting that HMGB1 acts as a key factor in the development of esophageal cancer. An shRNA targeting HMGB1 was designed for the knockdown of HMGB1 in ECA109 and TE13 cells, and the transfection efficiency of the shRNA was assessed using quantitative real-time reverse transcription polymerase chain reaction and western blot analysis. CCK-8 and clonogenic assays were used to analyze the effect of HMGB1 on the proliferation and radiosensitivity, respectively, of esophageal cancer cells in vitro. The influence of HMGB1 on radiation-induced changes in the migration, invasion, and cell cycle as well as apoptosis of tumor cells was examined by wound-healing and Transwell assays and flow cytometry, respectively. In addition, xenograft tumor models were constructed to observe the effect of HMGB1 on tumor growth in vivo. The results of the study in vitro revealed that the proliferation of the HMGB1-shRNA group decreased after irradiation, and the radiation treatment reduced the tumor volume of the xenograft model which was more marked in HMGB1-shRNA group. Moreover, HMGB1 was involved in the phosphorylation of H2AX after irradiation, and HMGB1 knockdown blocked the cell cycle in the G(0)/G(1) phase and increased apoptosis. HMGB1 deficiency was also correlated with the upregulation of p16, Bax and caspase-9 and the downregulation of MMP-2, MMP-9, cyclin D1, CDK4, H2AX and Bcl-2. These data indicated that the overexpression of HMGB1 prior to treatment was correlated with poor clinical outcome in esophageal carcinoma and that knockdown HMGB1 expression in human esophageal cancer cell lines increased their radiosensitivity by allowing the induction of apoptosis and G(0)/G(1) arrest after exposure to radiation.