Abstract: Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Transplantation outcome is often compromised by a rapid re-infection of the graft. Several factors have been implicated in the increased severity of recurrence, including steroid-based immunosuppression. Evidence suggests that steroid boluses used to treat acute rejection are associated with an increase in HCV viral load and the severity of recurrence. Two possible mechanisms for a steroid-mediated effect on HCV viral loads can be postulated, the first being a direct effect of steroids on the virus by enhancing its replication. The second, an indirect effect due to the suppression of the HCV immune response, allows unrestricted HCV replication. To investigate the direct effect of steroids on HCV replication, dexamethasone (Dex) and prednisolone (Pred) were tested in an in vitro replicon model. HCV replication was assessed on the basis of luciferase reporter expression (luminescence) and HCV RNA (RT-PCR). At clinically relevant concentrations (1–10 nM), treatment with both Dex and Pred did not enhance, but resulted in a slight reduction of relative luciferase activity (HCV replication), which was independent of increased cellular protein content and reduced cell proliferation. This minor reduction of HCV replication was confirmed by RT-PCR showing more than 41% reduction in HCV RNA levels. In conclusion, despite clinical evidence that the use of steroids aggravates recurrence of HCV, our in vitro study suggests that there is no direct stimulatory effect of steroids on the replication of HCV. As such, the increased viral loads after high-dose steroid treatment are more likely due to a downregulation of the immune response. In such patients, a dampened immune response allows viruses like HCV to replicate free of immune-mediated killing of their host cells. When a change occurs, such as a tapering or an alteration of immunosuppressant drugs, the immune system reinitiates and vigorously attempts to control the virus, resulting in acceleration of liver damage. Therefore, either steroid avoidance or maintaining low levels, coupled with a slow tapering of corticosteroids, may be beneficial to HCV-infected transplantation recipients.