Gene therapy could provide curative therapies to many inherited monogenic liver diseases. Clinical trials have largely focused on adeno-associated viruses (AAV) for liver gene delivery. These vectors, however, are limited by small packaging size, capsid immune responses, and inability to re-dose. As an alternative, nonviral, hydrodynamic injection through vascular routes can successfully deliver plasmid DNA (pDNA) into mouse liver but has achieved limited success in large animal models.We explored hydrodynamic delivery of pDNA through the biliary system into the liver of pigs using ERCP, and a power injector to supply hydrodynamic force. Human Factor IX (hFIX), deficient in Hemophilia B, was used as a model gene therapy.Biliary hydrodynamic injection was well tolerated without significant changes in vital signs, liver enzymes, hematology, or histology. No off-target pDNA delivery to other organs was detected by PCR. Immunohistochemistry revealed that 50.19% of the liver stained positive for hFIX after hydrodynamic injection at 5.5 mg pDNA, with every hepatic lobule in all liver lobes demonstrating hFIX-expression. hFIX-positive hepatocytes were concentrated around the central vein, radiating outward across all 3 metabolic zones. Biliary hydrodynamic injection in pigs resulted in significantly higher transfection efficiency than mouse vascular hydrodynamic injection at matched pDNA per liver weight dose (32.7-51.9% vs 18.9%, p