The radioresistance of tumor cells remains a major cause of treatment failure in nasopharyngeal carcinoma (NPC). Recently, several reports have highlighted the importance of epigenetic changes in radiation-induced responses. Here, we investigated whether the demethylating agent 5-azacytidine (5-azaC) enhances the radiosensitivity of NPC cells. The NPC cell lines CNE2 and SUNE1 were treated with 1 μmol/L 5-azaC for 24 h before irradiation (IR); clonogenic survival was then assessed. Tumor growth was investigated in a mouse xenograft model in vivo. The apoptosis, cell cycle progression and DNA damage repair were examined using flow cytometry, immunofluorescent staining and western blotting. Promoter methylation and the expression of four genes epigenetically silenced during the development of NPC were evaluated by pyrosequencing and real-time PCR. We found that pretreatment with 5-azaC significantly decreased clonogenic survival after IR compared to IR alone; the sensitivity-enhancement ratio of 5-azaC was 1.4 and 1.2 for CNE2 and SUNE1 cells, respectively. The combined administration of 5-azaC and IR significantly inhibited tumor growth in the mouse xenograft model, and enhanced radiation-induced apoptosis in vitro compared to 5-azaC alone or IR alone. 5-AzaC also decreased promoter methylation and upregulated the expression of genes which are epigenetically silenced both in vitro and in vivo in NPC. Thus, 5-azaC enhance the radiosensitivity of both the CNE2 and SUNE1 cell lines, possibly by altering DNA methylation levels and increasing the ability of irradiated cells to undergo apoptosis. The use of 5-azaC combined with IR maybe represent an attractive strategy for the treatment of NPC.