Human Phospholipid scramblase 1 (PLSCR1) is an α/β interferon-inducible protein that mediates antiviral activity against RNA viruses including vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). In the present study, we investigated the antiviral activity of PLSCR1 protein against HBV (Hepatitis B virus). Firstly, PLSCR1 mRNA and protein expression was found to be downregulated in HepG2 cells after HBV infection. Then by performing co-transient-transfection experiments in cells and hydrodynamics-based transfection experiments in mice using a HBV expression plasmid and a PLSCR1 expression plasmid, we found that PLSCR1 inhibited HBV replication in vitro and in vivo through a significant reduction in the synthesis of viral proteins, DNA replicative intermediates and HBV RNAs. We also demonstrated that the antiviral action of PLSCR1 against HBV occurs, partly at least, by activating the Jak/Stat pathway. In conclusion, our results suggest that the expression of PLSCR1 is involved in HBV replication and that PLSCR1 has antiviral activity against HBV.