Abstract: Objective To investigate the expression of human telomerase reverse transcriptase（hTERT）and hypermethylation of proteintyrosine phosphatase receptor（PTPRO）in human esophageal cancer cell line Eca-109 treated with norcantharidin（NCTD）. Methods Eca-109 cells were treated with different concentrations of NCTD（1，2，4，8，16 μg／ml）and the proliferation was determined by MTT assay at 12 h and 24 h. Cell apoptosis of NCTD+Eca-109 group（experimental group）and Eca-109 group（control group）were detected by TUNEL and laser scanning confocal microscope（LSCM）. Methylationspecific PCR（MSP）was used to detect the PTPRO methylation status in both experimental and control groups. hTERT protein was tested using Western blotting. Results MTT method showed that NCTD could increase the proliferation inhibitory rate of Eca-109 cells in a dose and time depended manner. The proliferation inhibitory rates of Eca-109 cells were statistically different among different concentrations of NCTD, as wall as different exposure time. Distinct morphological changes of apoptosis were found in experimental group tested by TUNEL and LSCM. PTPRO methylation status was found in control group by MSP test.The expression of hTERT protein was reduced in experimental group compared with control group（P＜0.05）. Conclusion NCTD can significantly inhibit the growth of esophageal cancer cell line Eca-109，which is associated with the down-regulation of hTERT and PTPRO methylation status.