Hepatitis C virus (HCV) entry is a multiple-step process involving a number of host factors and hence represents a promising target for new antiviral drug development. In search of novel inhibitors of HCV infection, we found that a human apolipoprotein E (apoE) peptide, hEP, containing both a receptor binding fragment and a lipid binding fragment of apoE specifically blocked the entry of cell culture grown HCV (HCVcc) at submicromolar concentrations. hEP caused little cytotoxicity in vitro and remained active even if left 24 hours in cell culture. Interestingly, hEP inhibited neither human immunodeficiency virus (HIV)-HCV pseudotypes (HCVpp) nor HIV and Dengue virus (DENV) infection. Further characterization mapped the anti-HCV activity to a 32-residue region that harbors the receptor binding domain of apoE, but this fragment must contain a cysteine residue at the N-terminus to mediate dimer formation. The anti-HCV activity of the peptide appears to be dependent on both its length and sequence and correlates with its ability to bind lipids. Finally, we demonstrated that the apoE-derived peptides directly blocked the binding of both HCVcc and patient serum-derived virus to hepatoma cells as well as primary human hepatocytes.