BACKGROUND & AIMS: An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and anti-viral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism dependent-viral characteristics or resistant mutations against anti-viral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH & RESULTS: We found that an 11 amino acid deletion (d11) in the preS1 region enhances the infectivity of cell culture-generated HBV (HBVcc) to sodium taurocholate co-transporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was approximately 10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pre-genomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the L-HBs protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture, and also for developing the anti-viral strategy against HBV infection.