Abstract: Objective：To establish the ρ° cells of the human esophageal carcinoma cells EC9706 and investigate the relationship between mtDNA copies and apoptosis. Methods ：Cells deficient mtDNA (ρ° cells) were acquired from ESCC cell lines EC9706 through continuous passage culture in the RPMI1640 supplemented with 50μg/ml EB, 50μg/ml uridine and 100μg/ml pyruvate,MtDNA copies of the two cell lines were detected at different time using the real-time fluorescence quantitative PCR after treated by EB. PCR products were validated by agarose gel electrophoresis; Apoptosis of ESCC cell lines EC9706 were analyzed using TUNEL staining and flow cytometry at different time after treated by EB. Results：The ρ°cells of ESCC cell lines EC9706 were successfully established. The Results：identified by the real-time fluorescence quantitative PCR indicated that mtDNA copies decreased progressively with the increasing in times of cell division in the presence of EB and mtDNA was disappear until 12 days.Apoptosis analysis was performed during the culture of ρ° cells of EC9706 after the cells were treated with EB on the 4th, 8th and 12th day. The Results： detected by flow cytometry indicated that apoptosis was increased gradually from the 4th day to 12th day. Apoptotic rates(%) were 2.78±1.04、11.68±1.85 and 26.62±1.06 in the cells EC9706. The apoptosis deteded by TUNEL was increased gradually from the 4th day to 12th day. Conclusion：Establishment of the ρ° cells of ESCC cell lines EC9706 offers new tools for research on the relationship between mitochondrial DNA and esophageal carcinoma.Apoptotic rate of EC9706 cells was increased gradually with the decrease of mtDNA copies. The Results： suggest that mtDNA may participate in the inducement of apoptosis and mtDNA lesions induced selectively could lead to mtDNA copies obvious decrease and further induce cell apoptosis. This is wished to become a new target for biotherapy of esophageal carcinoma.