Hepatitis C virus (HCV) chronically infects 3% of the world's population, and complications from HCV are the leading indication for liver transplantation. Given the need for better anti-HCV therapies, one strategy is to identify and target cellular cofactors of the virus lifecycle. Using a genome-wide siRNA library, we identified 96 human genes that support HCV replication, with a significant number of them being involved in vesicle organization and biogenesis. Phosphatidylinositol 4-kinase PI4KA and multiple subunits of the COPI vesicle coat complex were among the genes identified. Consistent with this, pharmacologic inhibitors of COPI and PI4KA blocked HCV replication. Targeting hepcidin, a peptide critical for iron homeostasis, also affected HCV replication, which may explain the known dysregulation of iron homeostasis in HCV infection. The host cofactors for HCV replication identified in this study should serve as a useful resource in delineating new targets for anti-HCV therapies.