BACKGROUND & AIMS: Cyclophilin inhibitors are being developed for treatment of hepatitis C virus (HCV) infection. They are believed to inhibit the HCV replication complex. We investigated whether cyclophilin inhibitors interact with interferon (IFN) signaling in cultured cells infected with HCV. METHODS: We used immunoblot assays to compare expression of IFN-stimulated genes (ISGs) and of components of IFN signaling in HCV-infected and uninfected cells. RESULTS: Incubation with IFN alfa induced expression of ISGs in noninfected cells and, to a lesser extent, in HCV-infected cells; addition of the cyclophilin inhibitor SCY-635 restored expression of ISG products in HCV-infected cells. SCY-635 reduced phosphorylation of double-strand RNA-dependent protein kinase (PKR) and its downstream factor eIF2 alpha; the phosphorylated forms of these proteins are negative regulators of ISG translation. Cyclophilin A interacted physically with PKR; this interaction was disrupted by SCY-635. SCY-635 also suppressed PKR-mediated formation of stress granules. Cyclophilin inhibitors were found to inhibit PKR phosphorylation and stress granule formation in HCV-infected and uninfected cells. CONCLUSIONS: In cultured cells, cyclophilin inhibitors reverse the attenuation of the IFN response by HCV, in addition to their effects on HCV replication complex. Cyclophilin A regulation of PKR has been proposed as a mechanism for observed effects of cyclophilin inhibitors on IFN signaling. We found that cyclophilin inhibitors reduce phosphorylation of PKR and eIF2 alpha during HCV infection to allow for translation of ISG products. Proteins in this pathway might be developed as targets for treatment of HCV infection.