OBJECTIVE To investigate the influence of combining RNAi-hTR plus hTERT genes upon the telomerase activity of bladder cancer BIU-87 cell line and provide new methods and evidence for RNAi in gene therapy of bladder transitional cell cancer. METHODS Three hTR-specific double-stranded siRNAs and 3 hTERT-specific double-stranded siRNAs were designed targeting different regions of hTR and hTERT mRNA. siRNAs (systems-PhTR-siRNA, PhTERT-siRNA and combining systems-PhTR plus PhTERT-siRNA) were transfected into bladder transitional cancer BIU-87 cell line. And hTR and hTERT mRNA expression were determined by fluorescence quantitative RT-PCR while telomeric repeat amplification protocol (TRAP) was applied to detect the telomerase activity and the growth inhibition of BIU-87 cells detected by MTT assay. RESULTS RNAi-pRNAT-hTERT-III, RNAi-pRNAT-hTR-III and combining RNAi-hTR plus hTERT could inhibit the expression of hTERT and hTR mRNA in bladder cancer BIU-87 cell lines by RNAi-pRNAT-hTERT-III hTERTmRNA 67%, RNAi-pRNAT-hTR-III hTRmRNA 41% and pRNAT-hTR-III hTRmRNA:57%, pRNAT-hTERT-III, pRNAT-hTR-III hTERTmRNA:70% (P < 0.05). The growth of bladder cancer BIU-87 cell was inhibited and telomerase activity considerably decreased, especially in combining RNAi-hTR and hTERT. CONCLUSIONS hTR-siRNA, hTERT-siRNA and combing siRNA hTR plus hTERT have been successfully designed and constructed. They can suppress specifically and effectively both hTR and hTERT mRNA expression and telomerase activity, especially in combining siRNA-hTR+hTERT. Combining siRNA-hTR plus hTERT are needed to explore its clinical applications.