Functional restoration of abdominal wall defects represents one of the fundamental challenges of reconstructive surgery. Synthetic grafts or crosslinked animal-derived biological grafts are characterized by significant adverse reactions, which are mostly observed after their implantation. The aim of this study was to evaluate the efficacy of the decellularization protocol to produce a completely acellular full-thickness abdominal wall scaffold. Full-thickness abdominal wall samples were harvested from Wistar rats and submitted to a three-cycle decellularization process. Histological, biochemical, and DNA quantification analyses were applied to evaluate the effect of the decellularization protocol. Mechanical testing and immunogenicity assessment were also performed. Histological, biochemical, and DNA analysis results showed efficient decellularization of the abdominal wall samples after the third cycle. Decellularized abdominal wall scaffolds were characterized by good biochemical and mechanical properties. The data presented herein confirm the effective production of a rat-derived full-thickness abdominal wall scaffold. Expanding this approach will allow the exploitation of the capacity of the proposed decellularization protocol in producing acellular abdominal wall scaffolds from larger animal models or human cadaveric donors. In this way, the utility of biological scaffolds with preserved in vivo remodeling properties may be one step closer to its application in clinical studies.