In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon-β (IFNB1) induction. Using a comprehensive microscopy-based high-content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A-interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF-κB p65 upon Sendai virus (SeV) infection. Notably, importin β1 (IMPβ1) knockdown-a hub protein highly targeted by several viruses-decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus-mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPβ1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPβ1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF-κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad-spectrum antiviral therapies.