Objective: To explore the cleaving and inhibitory activity of hepatitis C virus (HCV)-specific deoxyribozymes (DRz) at both molecular and transgeneic cellular levels. Methods: According to the secondary structure of HCV 5′-noncoding region (5′-NCR) and the sites characterized with 5′…Y↓R...3′(Y=A/G,R=U/C), HCV-specific naive deoxyribozymes were designed and named DRz-232, DRz-127, DRz-84, DRz1, and the phosphorothioate deoxyribozymes (PSDRz) and mutated phosphorothioate deoxyribozymes (MPSDRz) were also designed. HCV RNA 5′-NCR was transcribed in vitro from linearized plasmid pHCV-neo and radiolabelled at its 5′-end. DRz, PSDRz or MPSDRz was respectively mixed with the substrate RNA and incubated under appropriate conditions, the cleaved products were displayed by 8% denaturated polyacrylamide gel electrophoresis (PAGE) and autoradiography, and the optical density of each band was measured to calculate cleavage rates. After that, every kind of DRz was added respectively to the cultured transgeneic HepG2 cells containing luciferase gene controlled by HCV 5′-NCR. The cells were lysed at intended time points and the activity of luciferase was measured with chemiluminescence method for calculating inhibition rates. Results: After incubated for 90 min in vitro, the cleavage rates of DRz-127, PSDRz-127, DRz1 and PSDRz1 reached 32.6%, 30.8%, 24.3% and 21.5%, respectively. No cleavage product was observed in any MPSDRz. DRz-127, PSDRz-127, DRz1 and PSDRz1 had an inhibitory rate of 53.2%, 50.6%, 44.7% and 43.3% respectively in transgeneic HepG2 cells in the first 24 h when the final dose of the DRz was 0.5 μmol/L, higher than that of the corresponding MPSDRz. There was no significant difference between the inhibitory effect of each DRz and its PSDRz in HepG2 cells, but the inhibitory rate of DRz decreased more rapidly than that of the latter with the elapse of time. The results from transfection groups were significantly better than those of non-transfection groups. Conclusion: Rationally-designed HCV-specific deoxyribozymes are able to cleave target RNA at molecular level in vitro, and efficiently inhibit the expression of luciferase gene controlled by HCV 5′-NCR in transgeneic cells. Appropriate PSDRz may be more stable, and thus more suitable than the naive DRz in the application to cells. Introduction of the deoxyribozymes with transfection is more efficient than with direct delivering ways.