A variety of extracellular matrix (ECM) scaffolds have been promising in various tissue engineering applications, such as the porcine small intestinal submucosa. Yet no particular scaffolds have been optimal for vocal fold regeneration. The decellularized human umbilical vein (HUV) is a novel allogeneic scaffold that has shown some promise for cardiovascular tissue engineering. This study examines the potential of the HUV as an acellular scaffold for engineering the vocal fold, in order to develop an implantable tissue substitute that can promote constructive tissue remodeling. A saline-based decellularization protocol was used to fabricate a biodegradable, acellular, three-dimensional scaffold from native HUV tissue. Histological examination and scanning electron microscopy demonstrated that native cellular materials in the HUV were removed with a fine three-dimensional structure of proteins and proteoglycans well preserved. Primary human vocal fold fibroblasts were seeded on the abluminal surface of the acellular HUV scaffold and cultured for 21 days. Significant proliferation and infiltration of the fibroblasts in the scaffold were observed. These data supported the promise of the HUV scaffold for vocal fold regeneration and restoration.