Anterior tibial muscle of C57Bl6/J mice was subjected to decellularization with hypotonicsolution, detergents and DNase. Resulting acellular scaffolds were examined to characterize thecontent of chromatin, cell cytoplasm and extracellular matrix components incl. basal laminas,fibres and glycoproteins. Although the sarcoplasm and cell nuclei were removed, the generalskeletal muscle microarchitecture with ECM of stromal components remained well preserved atlight and electron microscopic levels. Moreover, basal laminas contouring honeycomb-likestructures left after removal of myofibres and vascular endothelium remained intact.Immunostaining of scaffolds for collagen IV and laminin confirmed positivity of basal laminas.Histochemical staining of deparaffinised scaffold sections identified well organized fibres afterstaining with green trichrome, Sirius red, Weigert’s resorcin fuchsin and Gomori impregnation.Chemical analysis gave evidence of reduced dsDNA and well-preserved collagen according tohigh hydroxyproline content and laminin as documented by Western blotting. We culturedscaffolds seeded with murine myogenic cells in vitro and confirmed their cytocompatibility asthe cells were able to adhere, grow and migrate through the ECM without affecting the scaffoldstructure. Myogenic cells were able to migrate in the endomysium and start to fuse.Implantation of decellularized scaffolds into an artificial cavity inside of anterior tibial muscle ofmice in vivo confirmed the scaffolds were colonized soon by recipient inflammatory cellswithout formation of foreign body giant cells. Scaffolds were well integrated with recipientskeletal muscle and gradually resorbed within 3 weeks. Our results confirm decellularized musclescaffold is a promising alternative for rebuilding the skeletal muscle organ as it can preservebasic chemical components and the tissue microstructure and show biocompatibility formyogenic cells as demonstrated in vitro and in vivo.