The interferon‐stimulated gene, viperin, has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here, we demonstrate that viperin plays an integral part in the ability of interferon to limit the replication of cell‐culture–derived HCV (JFH‐1) that accurately reflects the complete viral life cycle. Using confocal microscopy and fluorescence resonance energy transfer (FRET) analysis, we demonstrate that viperin localizes and interacts with HCV nonstructural protein 5A (NS5A) at the lipid‐droplet (LD) interface. In addition, viperin also associates with NS5A and the proviral cellular factor, human vesicle‐associated membrane protein‐associated protein subtype A (VAP‐A), at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C‐terminus, as well as an N‐terminal amphipathic helix. Removal of the amphipathic helix‐redirected viperin from the cytosolic face of the endoplasmic reticulum and the LD to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C‐terminal viperin mutants still localized to the LD interface and replication complexes, but did not interact with NS5A proteins, as determined by FRET analysis. Conclusion: In conclusion, we propose that viperin interacts with NS5A and the host factor, VAP‐A, to limit HCV replication at the replication complex. This highlights the complexity of the host control of viral replication by interferon‐stimulated gene expression. (HEPATOLOGY 2011;)