Short synthetic hairpin RNAs (sshRNAs) (SG220 and SG273) that target the internal ribosome entry site of the hepatitis C virus (HCV) were formulated into lipid nanoparticles and administered intravenously to HCV-infected urokinase plasminogen activator–severe combined immunodeficient mice with livers repopulated with human hepatocytes (humanized livers). Weekly administration of 2.5 mg/kg of each sshRNA for 2 weeks resulted in a maximal mean reduction in viral load of 2.5 log₁₀ from baseline. The viral load remained reduced by more than 90% at 14 days after the last dose was given. The sshRNAs were well tolerated and did not significantly increase liver enzyme levels. These findings indicate the in vivo efficacy of a synthetic RNA inhibitor against the HCV genome in reducing HCV infection.