Ionizing radiation represents one of the most important therapies for glioma, a lethal primary brain tumor, while radiotherapy remains a challenge for radiation oncologist because of radioresistance. Radiosensitivity of gliomas determines radiotherapy efficacy. Evidence demonstrated that methylation of CpG Island in the promoter region may result in gene silencing. This study was designed to determine the relationship between methylation status of ERCC1 promoter region and radiosensitivity in glioma cell lines. We investigated the expression levels of ERCC1 transcripts and protein in GBM cell lines. Colony forming experiments was used to measure surviving fraction at 2 Gy (SF2) in four human glioma cell lines, MGR1, MGR2, SF767 and T98G. Methylation status in the promoter region of ERCC1 in these glioma cell lines was determined by using bisulphate sequencing and MSP analysis. Radiosensitivity was examined to be heterogeneous in these glioma cell lines. There was a statistical difference in the radiosensitivity between glioma cell lines with and without methylation of ERCC1 gene promoter CpG islands. Furthermore, we promoted ERCC1 expression by 5-azacytidine treatment which resulted in the reduction of radiation-induced cell killing in radiosensitive cell lines. Our data indicate that methylation status of ERCC1 is associated with radiosensitivity in glioma cell lines. It could be used as a new biomarker for predicting the radiosensitivity of human gliomas.