Whole organ decellularization is a cell removal process that creates a natural extracellular matrix for use in transplantation. A lack of an intact endothelial layer in the vascular network of decellularized organs results in blood clotting even with anti-coagulation treatment. Furthermore, shear stress caused by blood flow may affect reseeded parenchymal cells. We hypothesized that a heparin-gelatin mixture (HG) can act as an antithrombotic coating reagent and induce attachment and migration of endothelial cells (ECs) on vascular wall surfaces within decellularized livers, with subsequent parenchymal cell function enhancement. Portal vein (PV) perfusion was performed for right lateral lobe decellularization of porcine livers. We tested if HG-precoating of isolated decellularized PV could increase EC attachment and migration. Additionally, we coated PV and hepatic artery walls in decellularized liver with HG, and then repopulated it with ECs and maintained it under vascular flow in a bioreactor for 10 days. Re-endothelialized scaffolds were perfused with porcine blood for thrombogenicity evaluation. We then co-cultured hepatocellular carcinoma (HepG2) cells and ECs to evaluate the effect of endothelialization on parenchymal cells. Finally, we transplanted these scaffolds heterotopically in pigs. HG improved ECs’ ability to migrate and adhere to vessel discs. ECs efficiently covered the vascular compartments within decellularized scaffolds and maintained function and proliferation after HG-precoating. No thrombosis was observed after 24 h blood perfusion in HG-precoated scaffolds, indicating an efficiently endothelialized vascular tree. HepG2 cells displayed a higher function in scaffolds endothelialized after HG-precoating compared to uncoated scaffolds in vitro and after in vivo transplantation. Our results lay the groundwork for engineering human-sized whole-liver scaffolds for clinical applications. Statement of Significance A major obstacle to successful organ bioengineering is vasculature reconstruction to avoid thrombosis and deliver nutrients through blood to the whole scaffold after in vivo transplantation. Although many attempts have been made to construct endothelial cell layers on the vascular network within decellularized organs, complete coverage has not be achieved. Here, we describe an effective approach for endothelial cell seeding to reconstruct a patent vascular tree within decellularized livers by coating the vasculature using heparin-gelatin mixture. Our results have demonstrate that enhancement of endothelial cell attachment by heparin-gelatin treatment could improve vascular patency and parenchymal cell function in vitro and in vivo . These results represent a significant advancement toward bioengineering functional liver tissue that maintains vascular patency for transplantation. [ABSTRACT FROM AUTHOR]