We tested the capsid targeted viral inactivation method as an anti-HBV strategy. HepG2 cells were cotransfected with HBV expression plasmid and the plasmid encoding fusion protein of either Core-A3C or Core-humanized renilla GFP (hrGFP). Core-A3C had substantial effect on HBV DNA levels. In the HepG2 cells expressing Core-A3C, the number of G-to-A mutations increased dramatically, whereas other nucleotide substitutions were rare. In addition, Core-A3C substantially inhibited HBV production intracellularly and in culture supernatant. These results suggest that Core-A3C may be a candidate as a novel antiviral agent against human HBV infection.