Allogenic hepatocyte transplantation or autologous transplantation of genetically modified hepatocytes has been used successfully to correct congenital or acquired liver diseases and can be considered as an alternative to orthotopic liver transplantation. However, hepatocytes are neither easily maintained in culture nor efficiently genetically modified and are very sensitive to dissociation before their reimplantation into the recipient. These difficulties have greatly limited the use of an ex vivo approach in clinical trials. In the present study, we have shown that primary human and rat hepatocytes can be efficiently transduced with a FLAP lentiviral vector without the need for plating and culture. Efficient transduction of nonadherent primary hepatocytes was achieved with a short period of contact with vector particles, without modifying hepatocyte viability, and using reduced amounts of vector. We also showed that the presence of the DNA FLAP in the vector construct was essential to reach high levels of transduction. Moreover, transplanted into uPA/SCID mouse liver, lentivirally transduced primary human hepatocytes extensively repopulated their liver and maintained a differentiated and functional phenotype as assessed by the stable detection of human albumin and antitrypsin in the serum of the animals for months. In conclusion, the use of FLAP lentiviral vectors allows, in a short period of time, a high transduction efficiency of human functional and reimplantable hepatocytes. This work therefore opens new perspectives for the development of human clinical trials based on liver-directed ex vivo gene therapy.