Background: The difficulties in eradicating hepatitis C virus (HCV) infection are mainly due to limited treatment options against the virus. Cyclosporine A (CsA) is an anti-inflammatory and immunosuppressive drug that has been widely used in organ transplantation and for treatment of various inflammatory diseases. We have recently reported that CsA specifically suppress HCV replication in vitro (BBRC 2004; 1:42). However, little has been understood about the effect of CsA on HCV replication and mechanism of its clinical effectiveness. In the present study, we have analyzed the mechanism of action of CsA on HCV replication. Materials and Methods: An HCV-1b or 2a replicon expressing chimeric firefly luciferase reporter and neomycin phosphotransferase (Feo) genes were stably transfected into human hepatoma Huh7 cells (Huh7/Rep-Feo). The Huh7/Rep-Feo cells were cultured in the presence of various concentrations of CsA. The level of intracellular HCV replication was quantified by luciferase assay. Expressions of cyclophilin genes were differentially suppressed by vector-derived shRNA directed against cyclophilins A, B, and C. Results: Treatment of Huh7/Rep-Feo with CsA significantly suppressed expression of the replicon by dose-dependent manner with a 50% inhibitory concentration of ฯณ0.5 g/ml. There was no change in the cell growth and viability, suggesting that the effect of CsA is due to specific suppression of HCV replication and not due to the cytotoxicity. In Western and Northern blot analyses the levels of both replicon RNA and HCV proteins were reduced by CsA treatment. Dose-effect analyses showed that CsA suppressed both HCV-1b and 2a replication to the same extents. The reporter assay showed that CsA did not activate ISRE-promoter nor had any effects on NFAT-promoter activities, suggesting that the antivirus action of CsA is independent of interferon-stimulated gene responses or CsA-calcineurin-NFAT mediated pathway. Contrary to CsA, another immune suppressive drug, FK506, did not suppress HCV replication. Furthermore, transcriptional knock down of cyclophilin A,B and C in Huh7/Rep-Feo cells resulted in significant decrease of the replicon expression. Taken together, the anti-HCV activity of cyclosporin A is mediated through specific blockade of cyclophilins. Conclusion: CsA substantially and specifically inhibits HCV replication in-vitro at clinically available concentrations, and the mechanisms of action may involve functional blockade cyclophilins. Further defining its points of action against HCV replication may potentially be important to locate novel molecular targets to terminate HCV replication.