This study evaluated a novel approach to decellularizing porcine adipose tissue while preserving its 3-D architecture. An ethanol-water mixture was used as a solvent to remove lipids and the number of freeze-thaw cycles (1-7), ethanol concentration, and tissue thickness were tested. Trypsin incubation time (1-3 h) and xylene immersion time were investigated separately. Processed sample microarchitecture was analyzed via scanning electron microscope, cellular content was analyzed via hematoxylin and eosin (H&E) staining, and DNA content was analyzed using gel electrophoresis. Tensile testing and five-stage incremental stress-relaxation testing was performed in phosphate-buffered saline at 37°C. Human neuroblasts were seeded and evaluated for infiltration and attachment over 8 days. Four cycles of freeze-thaw in 50% ethanol-water mixture removed one-third of the lipids. Microarchitecture showed the presence of pores, capillary channels, and lack of sidedness; H&E micrographs confirmed unaltered morphology and absence of cells. Incubation for 1.5 h in trypsin removed 99.5% DNA from delipidized samples. An average of 40% rehydration swelling, an elastic modulus of 324(±141) kPa, and an ultimate tensile strength of 87.4(±23.1) kPa were observed. The matrix exhibited strain hardening behavior similar to small intestinal submucosa. Cells successfully infiltrated and spread in the decellularized scaffold. Removal of lipids significantly reduced incubation in trypsin EDTA. In summary, the acellular matrix shows significant potential as a new template for tissue regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3127-3136, 2016.