Background: Restoring functionality to abdominal wall defects is a key challenge in reconstructive surgery. It is estimated that over 700,000 abdominal wall reconstructions are conducted annually in the United States, with more than 20 million performed worldwide each year. Synthetic grafts and crosslinked, animal-derived biological grafts often lead to significant adverse reactions following implantation. This study aimed to assess the effectiveness of a decellularization protocol in producing a fully acellular, full-thickness abdominal wall scaffold, an alternative therapeutic application for abdominal wall reconstruction. Methods: 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. In addition, implantation of decellularized abdominal wall scaffolds was performed at the scapular region of Sprague Dawley rats. The grafts remained for 4 weeks and were then explanted; histological analysis utilizing Hematoxylin and Eosin and immunohistochemistry against CD11b (macrophages), CD4 (T-helper cells), and CD8 (cytotoxic T cells) were performed to assess the biocompatibility potential. Results: 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. Biocompatibility assessment showed the successful migration of the host’s cells to the implanted abdominal wall scaffolds. Furthermore, no presence of CD11b, CD4, or CD8 cells was observed in the grafts after 4 weeks of implantation. Conclusion: The data presented herein confirm the effective production of a rat-derived, full-thickness abdominal wall scaffold. In addition, the scaffold was biocompatible after a 4-week implantation period. 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.