Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) regulates viral replication through its interaction with host and other viral proteins. We have previously shown that FK506-binding protein 8 (FKBP8) binds to NS5A and recruits Hsp90 to form a complex that participates in the replication of HCV. In this study, we examined the biochemical characteristics of the interaction and the intracellular localization of NS5A and FKBP8. Surface plasmon resonance analysis revealed that the dissociation constant of the interaction between the purified FKBP8 and NS5A expressed in bacteria was 82 nM. Mutational analyses of NS5A revealed that a single amino acid residue of Val or Ile at position 121, which is well conserved among all genotypes of HCV, is critical for the specific interaction with FKBP8. Substitution of the Val(121) to Ala drastically impaired the replication of HCV replicon cells, and the drug-resistant replicon cells emerging after drug selection were shown to have reverted to the original arrangement by replacing Ala(121) with Val. Examination of individual fields of the replicon cells by both fluorescence microscopy and electron microscopy (the correlative fluorescence microscopy-electron microscopy technique) revealed that FKBP8 is partially colocalized with NS5A in the cytoplasmic structure known as the membranous web. These results suggest that specific interaction of NS5A with FKBP8 in the cytoplasmic compartment plays a crucial role in the replication of HCV.