AbstractEx vivo gene manipulation in human hepatocytes is a promising therapeutic strategy in the treatment of inherited liver diseases. However, a major limitation is the lack of a highly efficient and safe genetic manipulation system for transplantable primary human hepatocytes (PHHs). Here, we reported that proliferating human hepatocytes (ProliHHs) cultured in vitro showed high susceptibility to lentivirus‐mediated genetic modification and maintained cellular phenotypes after lentiviral infection. Human factor VIII expression was introduced through F8‐Lentivirus‐mediated transduction of ProliHHs followed by xenotransplantation into immunocompromised haemophilia A mice. We demonstrated that these F8‐modified ProliHHs could effectively repopulate the mouse liver, resulting in therapeutic benefits in mouse models. Furthermore, no genotoxicity was detected in F8‐modified ProliHHs using lentiviral integration site analysis. Thus, this study demonstrated, for the first time, the feasibility and safety of lentiviral modification in ProliHHs to induce the expression of coagulation factor VIII in the treatment of haemophilia A.