Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is required for the replication of the viral genome and is involved in several host signaling pathways. To gain further insight into the functional role of NS5A in HCV replication, we screened human cDNA libraries by a yeast two-hybrid system using NS5A as the bait and identified human butyrate-induced transcript 1 (hB-ind1) as a novel NS5A-binding protein. Endogenously and exogenously expressed hB-ind1 was coimmunoprecipitated with NS5A of various genotypes through the coiled-coil domain of hB-ind1. The small interfering RNA (siRNA)-mediated knockdown of hB-ind1 in human hepatoma cell lines suppressed the replication of HCV RNA replicons and the production of infectious particles of HCV genotype 2a strain JFH1. Furthermore, these reductions were canceled by the expression of an siRNA-resistant hB-ind1 mutant. Among the NS5A-binding host proteins involved in HCV replication, hB-ind1 exhibited binding with FKBP8, and hB-ind1 interacted with Hsp90 through the FxxW motif in its N-terminal p23 homology domain. The impairment of the replication of HCV RNA replicons and of the production of infectious particles of JFH1 virus in the hB-ind1 knockdown cell lines was not reversed by the expression of an siRNA-resistant hB-ind1 mutant in which the FxxW motif was replaced by AxxA. These results suggest that hB-ind1 plays a crucial role in HCV RNA replication and the propagation of JFH1 virus through interaction with viral and host proteins.