Aberrant microRNA (miRNA) expression in cancer affects the transcription of target genes, and profoundly influences cancer-associated signaling pathways. Radiation resistance is a major problem encountered in the treatment of cancer. The present study aimed to investigate the role of miRNA (miR)-21 in the development of radiation resistance in non-small cell lung cancer cells. A radiation-resistant cell line was generated from A549 cells. Significant upregulation of miR-21 was detected in the radioresistant cancer cells, as compared with the radiosensitive cells, and overexpression of miR-21 rendered A549 parental cells resistant to radiation. In addition, glycolysis was increased in the radioresistant cells, as compared with the sensitive cells. Furthermore, hypoxia-inducible factor-1α (HIF1α) was upregulated by miR-21 in radioresistant cells, resulting in promotion of the key enzymes of glycolysis. Inhibition of HIF1α by small interfering RNA suppressed glycolysis and resensitized the cancer cells to radiation, whereas the recovery of HIF1α in miR-21-inhibited radioresistant cells resulted in recovery of radioresistance. In conclusion, the present study suggested that miR-21 may modulate radioresistance through the upregulation of HIF1α. These results may provide a novel perspective on miRNA for the development of anti-radioresistance drugs.