Tubular nerve guides have shown a potential to bridge nerve defects, by directing neuronal elongation, localizing growth factors, and inhibiting fibrotic cellular ingrowth. These investigations describe a novel acellular scaffold derived from the human umbilical cord artery that aims to enhance nerve regeneration by presenting a unique mechanical and chemical environment to the damaged nerve ends. A rapid, semiautomated dissection technique is described that isolates the human umbilical artery (HUA) from the umbilical cord, after which the vessel is decellularized using sodium dodecyl sulfate (SDS). The artery is turned inside out to produce a 3D scaffold, that unlike previous vessels for nerve repair, is more resistant to collapse. The scaffold has the potential as either an acellular bridge-implant, or for in vitro nerve regeneration. Stress–strain relationships and suture retention were assessed to determine whether the material had similar mechanical properties to native nerves. A dual process-flow perfusion bioreactor was developed to assess glucose mass transfer, and to investigate the culture of neuronal-like PC12 cells within the scaffold. These investigations have shown the automated dissecting method yields a smooth tubular scaffold, where wall thickness can be tuned to alter the mechanical behavior of the scaffold. Inverting the scaffold prevents collapse, with the decellularized iHUA having comparable mechanical properties to native nerves. Bioreactor cultures with PC12 cells seeded within iHUA lumenal void were shown to adhere and migrate into the preexisting ECM after 11 days of culture. These investigations show the potential of the iHUA as a unique 3D scaffold that may enhance nerve regeneration.