Numerous scaffold formulations have been investigated to support the regeneration of nucleus pulposus (NP) tissue for use as an early-stage therapy for intervertebral disc degeneration. Particular attention has focused on recreating the biochemical and mechanical properties of the native NP via the incorporation of exogenous extracellular matrix (ECM) components or synthetic surrogates. In the present study, we describe a novel approach to develop a tissue engineering (TE) scaffold comprised acellular porcine NP ECM. Complete decellularization of porcine NP was successfully achieved using a combination of chemical detergents, ultrasonication, and treatment with nucleases. Resulting NP scaffolds were devoid of host-cell remnants and the porcine antigen alpha-Gal. Native NP ECM components including aggrecan/chondroitin-6-sulfate and collagens types II, IX, and XI were found in physiologically relevant ratios within the NP scaffold. NP scaffold swelling capacity and unconfined mechanical properties were not significantly different from porcine NP tissue. Furthermore, NP scaffolds were conducive to repopulation with human adipose-derived stem cells as cell viability and proliferative capacity were maintained. These results demonstrate the successful decellularization of porcine NP and the resultant formation of a biomimetic scaffold exhibiting potential utility for TE the human NP.