To investigate the radiosensitivity effect of radioresistant nasopharyngeal carcinoma cell line CNE ‐ 2R after overexpressing miR‐486‐5p. Methods microRNA(miRNA) sequencing and RT‐qPCR were used to detect the expression level of miR‐ 486‐5p in the parent cell line CNE2 and the radioresistant cell line CNE‐2R. CNE‐2R cells were infected with miR‐486‐5p overexpress‐ ion and negative control lentivirus, which were defined as CNE‐2R‐Mimics group and CNE‐2R‐NC group, respectively. CCK‐8 assay and Clonogenic assay were used to detect the proliferation ability and the viability of cells irradiated with different dose of (0 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy) X‐ray. The cell apoptosis was detected by Annexin V‐APC/7‐AAD double stain assay. Results miRNA sequencing and RT ‐qPCR results showed that the level of miR ‐486‐5p in cell line CNE ‐2R was significantly lower than that in parent cell line CNE2. CCK‐8 assay revealed that the cell proliferation of CNE‐2R cells overexpressing miR‐486‐5p in CNE‐2R‐Mimics group was decreased compared with that in the CNE‐2R‐NC group after 48 h, 72 h and 96 h culture (all P<0.05). After the cells were irradiated with 2 Gy, 4 Gy, 6 Gy and 8 Gy irradiation, the survival fraction of CNE‐2R‐Mimics group was lower than that of the CNE‐2R‐NC group (all P<0.05), with the largest difference in the 8 Gy dose (t=30.152, P<0.001). Flow cytometry analysis indicated that the apoptosis rate of the CNE‐2R‐Mimics group was higher than that of CNE‐2R‐NC group at the irradiation dose of 0 Gy and 8 Gy (all P<0.05), and the difference of apoptosis rate under the irradiation dose of 8 Gy was more significant (t=14.945, P<0.001). The Clonogenic assay revealed that the clone formation fraction of the CNE‐2R‐Mimics group was lower than that of the CNE‐2R‐NC group under different doses, in which the radiobiological parameters D0, Dq, and the survival fraction in irradiation of 2 Gy in CNE‐2R‐Mimics group were lower than that of the CNE‐2R‐NC group (all P<0.05). Conclusions The nasopharyngeal carcinoma radioresistant cell line CNE‐2R overexpressing miR‐486‐5p may increase radiosensitivity by inhibiting cell proliferation and promoting apoptosis. [ABSTRACT FROM AUTHOR]