Abstract: Objective To explore the mechanism of high mobility group protein box 1 (HMGB1) in regulating the radio-sensitivity of non-small cell lung cancer (NSCLC). Methods The sensitivity of non-small cell lung cancer cells was detected by cloning assay. Western blot was performed to investigate the expression of HMGB1 and zinc finger E-box-binding protein 1 (ZEB1). The regulatory mechanism was investigated by double luciferase report and chromatin immunoprecipitation (ChIP) tests. Results According to Western blot analysis, HMGB1 regulated the expression of ZEB1. HMGB1 inhibited the radio-sensitivity of non-small cell lung cancer through targeting ZEB1, as confirmed by the double luciferase report and ChIP tests. Conclusions HMGB1 can regulate the radio-sensitivity of non-small cell lung cancer through targeting ZEB1, which provides theoretical basis for the clinical treatment of lung cancer.