RNA interference(RNAi) is the process of sequence-specific posttranscriptional gene silencing initiated by double-stranded RNA that is homologous to the silenced gene. To investigate the inhibitory effect of endogenous siRNA on target gene, a DNA-based vector was constructed for intracellular transcription of a hTERT specific short hairpin RNA(shRNA) and evaluate the silencing effect for hTERT gene expression. hTERT cDNA sequences from nucleotides 3565～3583 followed by 9 bp to form a loop and the corresponding antisense hTERT nucleotides followed by five thymines were cloned to the downstream of the U6 promoter in pPUR/U6 vector, resulting in the RNA interference vector pPUR/U6/hTERT. The constructed vector pPUR/U6/hTERT and the vancant vector pPUR/U6 were transferred into HepG2 cells respectively by Liposome-mediated transfection. The antibiotic-resistant transfected cells were selected and enriched by applying puromycin in culture medium. The surviving cells were seeded in 12-well plate for assaying cell growth rate by cell counting or harvested for evaluation of hTERT gene expression by RT-PCR and Western-blot assay. Telomerase activity was determined by TRAP(Telomerase Repeat Amplification Protocol) and p53 expression level was assayed by Western-blot. Compared to the cells transfected with pPURU6 control vector, pPUR/U6/hTERT caused efficient down-regulation of hTERT gene expression and telomerase activity. It also significantly increased p53 expression level and inhibited cell growth. These results suggested that the endogenous hairpin siRNA producted by DNA plasmid is capable of mediating robust hTERT gene inhibition. It is promised to be a potential tool for gene function analysis.