Hepatitis C virus (HCV) is highly dependent on host proteins for its own propagation. By transcriptome sequencing (RNA-Seq) analysis, we identified 30 host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these candidate genes, we selected and characterized ankyrin repeat domain 1 (ANKRD1). Here, we showed that protein expression of ANKRD1 was up-regulated in HCVcc-infected cells. We further showed that protein expression level of ANKRD1 was increased by nonstructural 5A (NS5A) protein. ANKRD1 specifically interacted with NS5A both in vitro and coimmunoprecipitation assays. Protein interaction was mediated through the domain II of NS5A and the C-terminal region of ANKRD1. Promoter activity of ANKRD1 was also increased by NS5A protein. Moreover, up-regulation of ANKRD1 expression was mediated through alteration in intracellular calcium homeostasis and ER stress in HCVcc-infected cells. We showed that silencing of ANKRD1 impaired HCV propagation without affecting HCV replication. By using HCV-like infectious particle (HCV-LP), we demonstrated that HCV single-cycle infection was drastically impaired in ANKRD1 knockdown cells. Finally, we verified that ANKRD1 was required for HCV entry. These data suggest that HCV coopts ANKRD1 for its own propagation and up-regulation of ANKRD1 may contribute to HCV-mediated liver pathogenesis.