Background: All To enhance the curative effect of radiotherapy, we established a radio-resistant cell line, CNE-2R from CNE-2, a radio-sensitive type of CNE, through repeated irradiation. The developed cell line provides a basis for further studies on the radio-resistance of CNE and the molecular mechanism of radiotherapy sensitization drugs. Materials and Methods: The CNE-2 cell line was selected and exposed repeatedly to high-dose X-ray intermittent irradiation (6 Gy/fraction for seven fractions and 20 Gy/fraction for three fractions). After the entire irradiation process, 20 Gy/time was administered to CNE irregularly. The cell growth curves of CNE-2 and CNE-2R were constructed based on MTT assays. Dose-survival curves were obtained through colony-forming tests and subjected to linear quadratic formulation matching. SF2 and correlation parameters of radiation biology were calculated. Changes in the cell cycle of CNE-2 and CNE-2R were also assessed by serum starvation. Results: The doubling time of CNE-2 was 2.4 days, which is 0.4 days shorter than that of CNE-2R, indicating the faster growth rate of CNE-2. In the dose-survival equation of the survival clone test, the sensitization ratio of CNE-2R was enhanced relative to that of CNE-2. After synchronization and desynchronization for 24 h, G1 was arrested in CNE-2R. In the S phase, which is insensitive to radiation, the ratio of G1 increased, hence altering the cell cycle. Conclusion: We have established a radio-resistant nasopharyngeal carcinoma cell line by repeated exposure to radiation which is relevant to changes in the cell cycle.