Diosgenin (3β-hydroxy-5-spirostene, 1), a plant-derived sapogenin, is used as a dietary supplement. However, the biological effects of 1 related to viral replication remain unexplored. In this study, the effects of 1 on hepatitis C virus (HCV) replication were evaluated. Based on a reporter-based HCV subgenomic replicon system, 1 was found to inhibit HCV replication at low micromolar concentrations. The EC(50) (concentration at which 50% of HCV replication is inhibited) of 1 was 3.8 μM. No cellular toxicity was observed at this concentration. Diosgenin (1) also significantly reduced the levels of viral RNA and viral proteins as evaluated by quantitative real-time reverse transcriptase PCR and Western blot analysis, respectively. In addition, in an alternative HCV antiviral system more closely aligned to all steps involved in the HCV infection and life cycle, 1 totally abolished HCV replication at 20 μM. Moreover, 1 reduced the phosphorylation of signal transducer and activator of transcription 3. A combination of 1 and interferon-α exerted an additive effect on the resultant anti-HCV activity.