Hepatitis C virus (HCV) envelope protein E2 is required for the entry of HCV into cells. Viral envelope proteins interact with cell receptors in a multistep process, which may be a promising target for the development of novel antiviral agents. In this study, a heptapeptide M13 phage-display library was screened for peptides that bind specifically to prokaryotically expressed, purified truncated HCV envelope protein E2. ELISA assay was used to quantify the binding of the peptides to HCV E2 protein. Flow cytometry, quantitative reverse-transcription PCR and western blotting were used to investigate the inhibition effect of one peptide on HCV infection in hepatoma cells (Huh7.5) in vitro. Four peptides capable of binding specifically to HCV E2 protein were obtained after three rounds of biopanning. Peptide C18 (WPWHNHR), with the highest affinity for binding HCV E2 protein, was synthesized. The results showed that peptide C18 inhibited the viral infectivity of both HCV pseudotype particles (HCVpp) harboring HCV envelope glycoproteins and cell-culture produced HCV (HCVcc). Thus, this study demonstrated that peptide C18 is a potential candidate for anti-HCV therapy as a novel viral entry inhibitor.