Abstract: To investigate the effect of 5-Azacytidine (5-Aza-C) on subtelomeric region D4Z4 repeats methylation, hTERT mRNA expression and telomerase activity in nasopharyngeal carcinoma (NPC) cell lines in vivo. Methods NPC cell lines CNE, CNE1, CNE2 and 5-8F were treated for 72h with 0, 1, or 2.5μmol/L of the demethylation agent 5-Aza-C. The methylation level of subtelomeric region D4Z4 repeats was evaluated with methylation-specific polymerase chain reaction(MSP); the human telomerase catalytic subunit (hTERT) mRNA expression was assayed by reverse transcription-polymerase chain reaction(RTPCR); the telomerase activity was detected using a telomeric repeat amplification protocol(TRAP) assay. Results Treatment with 5-Aza-C significantly decreased DNA methylation levels at the subtelomeric region D4Z4 repeats, and the degree of demethylation was statistically different from those before treatment. The telomerase activity was greatly reduced and the hTERT mRNA expression was strongly repressed in four NPC cells treated with 2.5μmol/L 5-Aza-C. Conclusion Abnormality of DNA methylation at the subtelomeric region plays a role in the development of NPC. 5-Aza-C could inhibit telomerase activity by downregulating hTERT mRNA expression in NPC cell lines. Detection of the association of hTERT expression with subtelomeric CpG methylation would provide better understanding of the role of subtelomeric structural chromosomal abnormality in NPC.