To explore the inhibitory effect in different positions siRNA on hTERT mRNA expression in MCF-7 cells at the different time points, we Designed and chemically synthesized four pairs of siRNA which aimed at human telomerase reverse transcriptase (hTERT) gene specifically, which were transfected into the MCF-7 cells by liposome method, and then the expression of hTERT mRNA in MCF-7 cells was tested respectively by half-quantity RT-PCR at 12 h, 24 h, 48 h, 72 h, and 5 d after transfection. Compared with the control groups, there were three pieces of siRNA that inhibit the expression of hTERT mRNA at 12 h after transfection among the four pieces. The highest inhibition ratio occurred at 48 h after transfection, and after 72 h the ratio descended, when the siRNA sequence was located at the relative simple structure site in the secondary structure of hTERT mRNA had the highest inhibition ratio, which was 75%(P<0.01), which indicated that the specific siRNA had obvious inhibition effect on hTERT gene, and the effect was dependant positionally and timely dependence.