A variety of xenogeneic and allogeneic 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 lamina propria, in order to develop an implantable tissue substitute that can promote a natural ECM remodeling response. A novel saline-based decellularization protocol developed in our laboratory was used to fabricate a threedimensional (3-D), biodegradable, acellular scaffold from native HUV tissue. Histological examination and scanning electron microscopy indicated that native cells in the HUV were removed with a fine 3-D structure of proteins and proteoglycans well preserved. Primary human vocal fold fibroblasts were cultivated on the abluminal surface of the acellular scaffold in vitro. Significant proliferation and infiltration of the fibroblasts in the scaffold were observed. These findings supported the biocompatibility of the HUV scaffold, and its promise for vocal fold reconstruction and regeneration.