HCV NS5A is a dimeric phosphoprotein involved in HCV replication. NS5A inhibitors are among direct‐acting antivirals (DAA) for HCV therapy. The Y93H mutant of NS5A is resistant to NS5A inhibitors, but the precise mechanism remains unclear. In this report, we proposed a Ser38‐His93‐Asn91 triad to dissect the mechanism. Using pymol 1.3 software, the homology structure of JFH1 NS5A was determined based on the dimer structure of genotype 1b extracted from the database Protein DataBank ( www.ebi.ac.uk/pdbsum ) with codes 1ZH1 and 3FQM / 3FQQ . FLAG‐NS5A‐WT failed to form dimer in the absence of nonstructural proteins from subgenomic replicon (NS3‐5A); however, FLAG‐NS5A‐Y93H was able to form dimer without the aid of NS3‐5A. The Ser38‐His93‐Asn91 triad in the dimer of the Y93H variant predicts a structural crash of the cleft receiving the NS5A inhibitor daclatasvir. The dimerization assay revealed that the existence of JFH1‐NS5A‐ 1ZH1 and ‐ 3FQM homology dimers depended on each other for existence and that both NS5A‐WT 1ZH1 and 3FQM dimers cooperated to facilitate RNA replication. However, NS5A‐Y93H 1ZH1 alone could form dimer and conduct RNA replication in the absence of the 3FQM structure. In conclusion, this study provides novel insight into the functional significance of the Ser38‐His93‐Asn91 triad in resistance of the Y93H variant to NS5A inhibitors.