We and others previously reported that the direct-acting agents (DAA) NS5A inhibitors (NS5Ai) and the host-targeting agents cyclophilin inhibitors (CypIs) inhibit HCV replication in vitro. In this study, we investigated whether the combination of NS5Ai and CypI offers a potent anti-HCV effect in vivo. A single administration of NS5Ai or CypI alone to HCV-infected humanized-mice inhibits HCV replication. The combination of NS5Ai with CypI suppresses HCV (GT1a, GT2a, GT3a and GT4a) replication in an additive manner. NS5Ai/CypI combinations provide a statistically more profound anti-HCV inhibition for GT2a and GT3a than GT1a and GT4a, leading to a fastest and deepest inhibition of GT2a and GT3a replications. Combining CypI with NS5Ai prevents the viral rebound normally observed in mice treated with NS5Ai alone. Results were confirmed in mice implanted with human hepatocytes from different donors. Therefore, the combination of NS5Ai with CypI may serve as a regimen for the treatment of HCV patients with specific genotypes and disorder conditions, which diminish sustain viral response levels to DAA, such as GT3a infection, cirrhosis, and DAA resistance associated with the selection of resistance-associated substitutions present at baseline or are acquired during treatment.