The kinase C-related kinase 2 (PRK2), which phosphorylates hepatitis C virus (HCV) RNA polymerase, is a proviral factor enhancing HCV replication. Here, we report on the in vivo anti-HCV efficacy of HA1077, which inhibits viral genome replication by targeting PRK2 and displays viral entry inhibitory activity by targeting Rho-associated kinase. HA1077 showed synergistic antiviral activity selectively with nonstructural protein 5 A (NS5A) inhibitors including daclatasvir (DCV). HA1077 oral administration substantially reduced serum viral loads in mice bearing HCV genotype 2a-replicating Huh7 xenografts. When administered with DCV, HA1077 potentiated the antiviral efficacy of DCV and suppressed the generation of DCV resistance-associated variants (RAVs). By deep-sequencing analysis, we uncovered an unprecedented DCV-induced polymorphism at the poly-proline motif (PxxPxxP) of NS5A. Coadministration of HA1077 reduced such a polymorphism. Overall, our results demonstrate the potential therapeutic benefit of combination therapy with HA1077 plus DCV for HCV patients carrying emerging or pre-existing RAVs toward NS5A inhibitors.