Abstract: Objective To study the effects of suppressing cell proliferation and hTERT gene expression on HeLa cells with ZD55-TERT-siRNA,an oncolyic adenovirus associated small interference RNA targeting hTERT gene. Methods ZD55-EGFP, an E1b-55kD gene-deleted oncolytic adenovirus, AD-TERT-siRNA, a replication-deficient adenovirus expressing hTERT-siRNA and AD-EGFP, a replication-deficient adenovirus were used as control groups. Human cervical carcinoma HeLa cells were infected by these four kinds of adenoviruses using different titers respectively. Cytotoxicity was tested by crystal violet staining. Cell proliferation was assayed by MTT method. hTERT expression levels of transfected HeLa cells were detected by RT-PCR,Western blot and immunocytochemistry respectively. The expression of E1A protein was examined by immunocytochemistry and Western blot. Results The cytotoxicity and inhibitory effects of ZD55-TERT-siRNA on HeLa cells were the most significant while ZD55-EGFP and AD-TERT-siRNA were more potent than AD-EGFP. Adenoviral particles could be detected in tumor cells infected by ZD55-TERT-siRNA and ZD55-EGFP，but not in the cells infected with AD-TERT-siRNA and AD-EGFP can not. The most significant inhibition of hTERT expression were observed in lysates from HeLa cells infected with ZD55-TERT-siRNA. Conclusion ZD55-TERT-iRNA has the most significant antitumor effect and can significantly knockdown hTERT expression.