Lentiviral vectors (LVs) are attractive vehicles for liverdirected gene therapy by virtue of their ability to stably integrate in the genome of target cells and the lack of pre-existing immunity against vector components in most humans. Over the past years, we have developed a LV platform that can achieve stable transgene expression in the liver and establish correction of hemophilia in mouse models upon systemic administration. This LV is designed to stringently target transgene expression to hepatocytes through transcriptional and microRNA-mediated regulation. We then investigated the efficacy and safety of portal vein administration of LVs expressing wild-type, codonoptimized (c.o.) or c.o. hyperfunctional canine factor IX (cFIX) in a canine model of hemophilia B. We observed long-term stable reconstitution of cFIX activity up to 1\% of normal and significant amelioration of the clinical phenotype in 3 treated dogs (10 years cumulative follow up). In the perspective of clinical translation and to increase therapeutic efficacy, we next treated a hemophilia B dog by peripheral vein administration of LVs expressing the c.o. hyperfunctional cFIX at a 5-fold higher dose than those previously administered. At the current followup (6 months after gene therapy) cFIX activity is 7–8\% of normal, suggesting comparable efficacy of LV by both portal and peripheral vein administration Treatment of more hemophilia B dogs is underway to extend these results. Overall our studies position LV-mediated liver gene therapy for further preclinical development and clinical translation. LVs may thus complement other available vectors for liver gene therapy of hemophilia.