To investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A).Yeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods.Among twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase.Genes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.