Human aci-reductone dioxygenase 1 (ADI1) is a member of the Cupin superfamily. It binds to and inhibits the activities of membrane-type 1 matrix metalloproteinase, a protein known to interact with the tight junction protein, claudin-1. Previously, a variant protein, named submergence-induced protein-like factor (Sip-L), consisting of ADI1 amino acids 64-179, was found to support hepatitis C virus (HCV) infection and replication in 293 cells. In the present study, it was discovered that over-expression of human ADI1 in 293 cells (293-ADI1 cells) also supported HCV infection and replication. Using serum-derived HCV as an infectious source, enhanced cell uptake of HCV to a Northern blot detectable level was found in 293 cells over-expressing both CD81 and ADI1 (293-ADI1-CD81 cells). The enhanced cell entry was confirmed by the use of the vesicular stomatitis virus-based HCV pseudotype particles. However, transfection of HCV replicon RNA by electroporation into naïve 293 and 293-ADI1 cells revealed no difference in replication efficiency. Using the infectious J6/JFH chimera as an infectious source, the infectivity was compared between 293-ADI1-CD81 and Huh-7.5 cells. More infection foci were formed in the 293-ADI1-CD81 cells in the first round of infection. In conclusion, human ADI1 over-expression in 293 cells enhances cell entry but not replication of HCV. 293-ADI1-CD81 cells are permissive for serum-derived HCV infection.