Nucleolin (C23) is an important anti-apoptotic protein that is ubiquitously expressed in exponentially growingeukaryotic cells. In order to understand the impact of C23 in radiation therapy, we attempted to investigate therelationship of C23 expression with the radiosensitivity of human non-small cell lung cancer (NSCLC) cells.We investigated the role of C23 in activating the catalytic subunit of DNA-dependent protein kinase (DNAPKcs),which is a critical protein for DNA double-strand breaks (DSBs) repair. As a result, we found that theexpression of C23 was negatively correlated with the radiosensitivity of NSCLC cell lines. In vitro clonogenicsurvival assays revealed that C23 knockdown increased the radiosensitivity of a human lung adenocarcinomacell line, potentially through the promotion of radiation-induced apoptosis and adjusting the cell cycle to a moreradiosensitive stage. Immunofluorescence data revealed an increasing quantity of γ-H2AX foci and decreasingradiation-induced DNA damage repair following knockdown of C23. To further clarify the mechanism of C23in DNA DSBs repair, we detected the expression of DNA-PKcs and C23 proteins in NSCLC cell lines. C23 mightparticipate in DNA DSBs repair for the reason that the expression of DNA-PKcs decreased at 30, 60, 120 and 360minutes after irradiation in C23 knockdown cells. Especially, the activity of DNA-PKcs phosphorylation sitesat the S2056 and T2609 was significantly suppressed. Therefore we concluded that C23 knockdown can inhibitDNA-PKcs phosphorylation activity at the S2056 and T2609 sites, thus reducing the radiation damage repairand increasing the radiosensitivity of NSCLC cells. Taken together, the inhibition of C23 expression was shownto increase the radiosensitivity of NSCLC cells, as implied by the relevance to the notably decreased DNA-PKcsphosphorylation activity at the S2056 and T2609 clusters. Further research on targeted C23 treatment maypromote effectiveness of radiotherapy and provide new targets for NSCLC patients.