Engineering three-dimensional tissues in vitro for regenerative therapy is highly desired. Vascular beds with connectable arteries and veins play crucial roles in fabricating three-dimensional tissues in vitro and improving the survival rate of transplanted three-dimensional tissues in vivo. Here, we developed a method to reconstruct a new vascular bed that could be implanted into humans for clinical applications. Porcine small intestine with an arteriovenous loop was selected as a vascular bed skeleton and decellularized using transvascular perfusion with both detergent and enzyme. Subsequently, the decellularized intestinal graft was re-endothelialized with human cells to allow blood perfusion into the vascular bed. The small intestinal graft was successfully decellularized without severe damage to the burst pressure of the graft, and the decellularized intestinal graft could be transplanted without a severe inflammatory response in rats for two weeks. In addition, the decellularized intestinal graft was partially vascularized with human endothelial cells in seven days. Therefore, the reconstructed intestinal vascular bed may serve as a human transplantable vascular bed with potential in clinical investigations.