The present paper aimed to find out the interacting protein of hepatitis E virus (HEV) capsid protein ORF2 and explore its role in HEV infection. CD63 was identified as a potential HEV ORF2-interacting protein by screening a human liver cDNA library for yeast two-hybrid method. Co-immunoprecipitation and pull-down assays were used to verify the interaction. The expression of cell surface CD63 was detected by flow cytometry in HEV susceptible, non-susceptible and overexpressed cells (PLC/PRF/5) respectively. HEV infectivity was measured through virus infection assay. The results indicated that expression level of CD63 on the cell membrane of HEV susceptible cell lines was lower than that of HEV non-susceptible cell lines. Overexpression of CD63 inhibited the HEV infection of PLC/PRF/5 cells, while siRNA interference of CD63 promoted the HEV infection. The results indicated that CD63 could interact with HEV ORF2, and may inhibit HEV infection in hepatocytes.