High mobility group box 1 (HMGB1) has been reported to regulate the sensitivity of several types of cancer cell to chemoradiotherapy. The present study aimed to investigate the changes in HMGB1 expression after radiotherapy, as well as its regulatory role in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. The expression levels of HMGB1 in the serum of 73 patients with NSCLC were analyzed by ELISA. HMGB1 mRNA and microRNA (miR)-107 expression in NSCLC cells were assessed using reverse transcription-quantitative PCR. Receiver operating characteristic analysis was used to evaluate the diagnostic value of HMGB1. Cell counting kit-8, Transwell invasion and clonogenic assays were used to determine cellular viability, invasiveness and colony formation ability, respectively. Following radiotherapy, the levels of HMGB1 were significantly decreased in the serum of patients with NSCLC, and lower serum levels had relatively high diagnostic accuracy in radiosensitive patients. Furthermore, HMGB1-knockdown retarded cellular proliferation and invasion with or without irradiation, and enhanced NSCLC cell radiosensitivity. Furthermore, knocking down miR-107 reversed the decreases in cellular proliferation and invasiveness both with and without irradiation, and reduced the survival fractions induced by sh-HMGB1. HMGB1-knockdown leads to radiosensitivity that may result from suppression of the Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. Collectively, decreased expression of HMGB1 was found to be a putative diagnostic predictor of radiosensitivity in patients with NSCLC. HMGB1-knockdown inhibited the proliferation and enhanced the radiosensitivity of NSCLC cells, which may be regulated via miR-107 by mediating the TLR4/NF-κB signaling pathway. Thus, HMGB1 may be a potential regulator of radioresistance in NSCLC, and the HMGB1/miR-107 axis may represent a promising therapeutic target.