Purpose: Radiotherapy is a major treatment method for patients with non-small cell lung cancer (NSCLC). However, the presence of radioresistant cancer stem cells (CSCs) may be associated with disease relapse or a poor outcome after radiotherapy. Voltage-gated calcium channel alpha 2 delta 1 subunit (encoded by the gene CACNA2D1) isoform 5 is a marker of CSCs in hepatocellular carcinoma. This study aimed to investigate the radiosensitivity of alpha 2 delta 1-high cells in NSCLC cell lines. Materials and methods: NSCLC cell lines A549, H1975, H1299, and PC9 were used. CACNA2D1-knockdown and CACNA2D1-overexpressing cell lines were established by lentiviral infection. Colony formation assay was performed to determine radiosensitivity. Sphere formation assay in serum-free medium was performed to evaluate self-renewal capacity. Proteins associated with DNA damage repair were analyzed by immunofluorescence or Western blot. The monoclonal antibody of alpha 2 delta 1 was applied alone or in combination with radiation either in vitro or in vivo to determine the anti-tumor effect of the antibody. Results: alpha 2 delta 1-high cells showed greater sphere-forming efficiency than alpha 2 delta 1-low cells and were relatively resistant to radiation. CACNA2D1 knockdown in A549 cells enhanced radiosensitivity, whereas CACNA2D1 overexpression in PC9 and H1975 cells reduced radiosensitivity, suggesting that alpha 2 delta 1 imparted radioresistance to NSCLC cells. Analysis of proteins involved in DNA damage repair suggested that alpha 2 delta 1 enhanced the efficiency of DNA damage repair. The monoclonal antibody of alpha 2 delta 1 had a synergistic effect with that of radiation to block the self-renewal of alpha 2 delta 1-high cells and enhanced the radiosensitivity of alpha 2 delta 1-positive cells in colony formation assays. The combination of the alpha 2 delta 1 antibody with radiation repressed A549 xenograft growth in vivo. Conclusion: alpha 2 delta 1 enhances radioresistance in cancer stem-like cells in NSCLC. The alpha 2 delta 1 monoclonal antibody sensitizes alpha 2 delta 1-high cells to radiation, suggesting that the antibody may he used to improve the treatment outcome when combined with radiation in NSCLC.