Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha, the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the characterization of two novel proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar in vitro activity against both genotype I and genotype II HCV. These proteins inhibit viral infectivity by binding to the HCV envelope glycoprotein E2 and blocking viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the 25 kDa protein griffithsin, comprised of domain-swapped homodimer of 12.7-kDa subunits, is bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model with engrafted human hepatocytes.