BACKGROUND AND AIMS: A simple, safe, targeted, and efficient in vivo DNA delivery system is necessary for clinical-grade liver-targeted gene therapy in humans. Intravascular hydrodynamic gene delivery has been investigated in large animal models, but translation to humans has been hampered by its technical challenges, invasiveness, and potential for significant cardiovascular adverse events. We posited that intrabiliary delivery of DNA plasmids via ERCP-guided hydrodynamic injection could overcome these obstacles. METHODS: Twelve pigs (40-50 kg) were divided into 3 groups (4 per group) and survived 21, 30, or 60 days. ERCP was performed by inflating a balloon catheter in the common hepatic duct and creating a closed space between it and the liver parenchyma. Last, a solution composed of plasmid/sleeping beauty (SB) mix was injected under pressure through the catheter into the closed space. Swine were killed at the 3 different time points and liver tissue harvested. Plasmid DNA expression and functional translated protein expression were assessed. RESULTS: ERCP-guided hydrodynamic delivery of naked plasmid DNA facilitated by pCytomegalovirus-Sleep Beauty (pCMV-SB) transposons was technically feasible and devoid of cardiovascular and local adverse events in all 12 pigs. Furthermore, plasmid DNA (both single and combination) was successfully transferred into swine hepatocytes in all 12 pigs. Additionally, stable integration of the DNA constructs in hepatocyte genomic DNA was reliably noted at all 3 time points. In the 4 swine that were kept alive to 60 days, successful genomic integration and subsequent protein expression was observed in the targeted liver tissue. CONCLUSIONS: ERCP-guided hydrodynamic delivery of gene therapy may usher in the next chapter in gene therapy with the potential to impact a variety of single-gene, complex genetic, and epigenetic liver diseases. It also raises the possibility that other nucleic acid therapeutics (microRNA, lncRNA, siRNA, shRNA) could similarly be delivered.