These investigations describe the development of a novel ex vivo three-dimensional scaffold derived from the human umbilical vein (HUV), and its potential as a regenerative matrix for tissue regeneration. Unique properties associated with the vascular wall have shown potential to function as a surgical barrier for guided tissue regeneration, particularly with the regeneration of periodontal tissues. HUV was isolated from umbilical cords using a semiautomated machining technology, decellularized using 1% sodium dodecyl sulfate, and then opened longitudinally to form tissue sheets. Uniaxial tensile testing, stress relaxation, and suture retention tests were performed on the acellular matrix to evaluate the HUV's biomechanical properties, followed by an evaluation of cellular interactions by seeding human gingival fibroblasts to assess adhesion, metabolic function, and proliferation on the scaffold. The scaffold's biomechanical properties were shown to display anisotropic behavior, which is attributed to the ex vivo material's composite structure. Detailed results indicated that the ultimate tensile strength of the longitudinal strips was significantly higher than that of the circumferential strips (p < 0.001). The HUV also exhibited significantly higher stress relaxation response in the longitudinal direction than in the circumferential orientation (p < 0.05). The ablumenal and lumenal surfaces of the material were also shown to differentially influence cell proliferation and metabolic activity, with both cellular functions significantly increased on the ablumenal surface (p < 0.05). Human gingival fibroblast migration into the scaffold was also influenced by the organization of extracellular matrix components, where the lumenal surface inhibits cell migration, acting as a barrier, while the ablumenal surface, which is proposed to interface with the wound site, promotes cellular invasion. These results show the HUV bioscaffold to be a promising naturally derived surgical barrier that may function well as a resorbable guided tissue regeneration membrane as well as in other clinical applications.