Objective   To investigate the antitumor effects of oncolytic adenovirus ZD55-double-hTERT expressing double small hairpin RNA targeting hTERT gene on prostate cancer DUl45 cells．Methods   The inhibitory effect of ZD55-doublehTERT on hTERT expression was evaluated by quantitative realtime PCR and Western blot analysis．The expression of E1A protein in DUl45 cell infected with viruses respectively was detected by Western blot，The cytotoxicity of ZD55-double-hTERT was detected by MTT assay．The apoptosis effect was determined by Hoechst-33342. Results   Real-time PCR and Western blot assay showed that ZD55doublehTERT could significantly decrease the expression of hTERT in DUl45 cells. Western blot assay of the E1A expression indicated DU145 cells infected with adenoviruses could express E1A, but blank group could not. MTT assay showed that cell viability of ZD55-double-hTERT group was significantly lower than that in other groups．ZD55-double-hTERT treatment of DU145 cells increased apoptotic cell death as compared to ZD55-hTERT1, ZD55-hTERT2 and blank group. Conclusions   Oncolytic adenovirus ZD55-double-hTERT can more effectively knock down the expression hTERT in DUl45 cells, and exert more dramatically antitumor effects than ZD55-hTERT1 and ZD55-hTERT2．