Whole rats’ diaphragms were decellularized with adetergent-enzymatic method using a newly designed bioreactor. We obtained a complete removal of all cellular components in the decellularized matrices while preserving the biomechanical properties and biological functions of the extracellular matrix (positive staining of collagen I, collagen IV, laminin, VEGF, elastin, fibronectin). The in vitro and in vivodata of thescaffolds proved non-immunogenic, biocompatible, non-toxic and evoke a low level of reactive oxygen species (ROS). Potential impact on seeded with mesenchymal stromal cells differentiation was investigated by gene expression and immunostaining. Using a transversal laparotomy, the entire left hemidiaphragm was resected and replaced with the bioengineered scaffold, and long-term (6 month) outcome observed. The orthotopic implantation showed excellent biocompatibility, neovascularization and intermediate comparable native-like functional characteristics (recovery of contractility, maintaining mobility) for the engineered diaphragm graft and in situ regeneration with positive staining for muscular and mesothelium cells.