Background: A combination of pegylated interferon and ribavirin is the current standard therapy for hepatitis C virus (HCV) infection, but this combination provides relatively low efficacy, especially in some patients with HCV genotype 1 infection; therefore, the development of novel therapeutic agents is required for further improvement in the treatment of chronic HCV infection. Methods: HCV pseudotype and subgenomic replicon assays were used in this study. The interaction of compounds with HCV receptors was examined using flow cytometry. Intracellular RNA levels were determined by semi-quantitative reverse transcriptase PCR. Results: Procyanidin B1 (PB1), a dimer of (-)- epicatechin and (+)-catechin, purified from Cinnamomi cortex, inhibits infection by vesicular stomatitis virus and HCV pseudotype virus in Huh-7 cells, with 50% effective concentrations of 29 and 15 μM, respectively. No inhibitory effects were observed in each component of PB1. We found that PB1 does not interfere with viral entry or receptor expression, but inhibits HCV RNA synthesis in a dose-dependent manner. Conclusions: These results indicate that PB1 suppresses HCV RNA synthesis, possibly as a HCV RNA polymerase inhibitor. Our results might contribute towards the development of more effective inhibitors for HCV infection from natural plants.