Abstract: Objective To study the inhibitory effect of antisense human telomerase reverse transcriptase (hTERT) on leukemia cell proliferation in vitro. Methods Sense and antisense hTERT eukaryotic expression vectors previously constructed were transfected into leukemia cell line HL₆₀ using SuperFect transfection reagent (Qiagen) to obtain HL₆₀-s and HL₆₀-as, and the G418-resistant colonies were identified for the presence of hTERT insert by PCR with T7 and pcDNA3.1/BGH reverse primers. Endogenous hTERT mRNA expression and telomerase activity were then detected by quantitative real-time RT-PCR and telomerase associated protein -silver staining in each cell line. MTT cellular proliferation assay, soft agar colony formation assay and flow cytometry were also employed to analyze the changes in proliferation capacity of leukemia cell in vitro and apoptosis of the tumor cells induced by antisense hTERT. Results Antisense hTERT remarkably reduced endogenous hTERT mRNA expression (P<0.01) and down-regulated telomerase activity in HL₆₀, as compared with the blank control and sense hTERT. After 25 passages of the 3 cell lines, a 7-day cell growth curve and the numbers (size) of soft agar colony formation showed that the proliferation rates and the anchorage-independent growth ability of HL₆₀-as cells were significantly decreased in comparison with HL₆₀ and HL₆₀-s cells, but a significant increase in apoptosis of HL₆₀-as cells occurred as determined by flow cytometry. Conclusions Antisense hTERT can obviously inhibit leukemia cell growth and proliferation in vitro, and this telomerase-targeted molecular biotherapy may be achieved by apoptosis pathway through down-regulation of hTERT mRNA and telomerase activity.