UNLABELLED: Muscle injury and defect affect people's quality of life, and effective treatment is lacking. Herein, we generated a scaffold to obtain decellularized porcine Achilles tendon myotendinous junction (D-MTJ) extracellular matrix (ECM) with well-preserved native biphasic hierarchical structure, biological composition, and excellent mechanical properties for muscle regeneration. The combined use of potassium chloride, potassium iodide, Triton-X 100, and sodium-dodecyl sulfate (SDS) can completely remove the main immunogenicity, while maintaining the major biological components and microstructure. The specific biomechanics of D-MTJ is comparable to the native muscle-tendon physiological conditions. Additionally, the D-MTJ ECM scaffold induced minimal immunological reaction (histology analysis) through rat subcutaneous implantation. Moreover, in vitro, muscle satellite cells adhered, proliferated, and infiltrated into the D-MTJ scaffold, and myofiber-like cell differentiation was observed as shown by increased expression of myogenesis-related genes during culture. In vivo, newly formed myofibers were observed in a muscle defect model with D-MTJ orthotopic transplantation, while the control group presented mostly with fibrous tissue deposition. Additionally, the number of Myod and MyHC-positive cells in the ECM scaffold group was higher at day 30. We preliminary explored the mechanisms underlying D-MTJ-mediated muscle regeneration, which may be attributed to its specific biphasic hierarchical structure, bio-components, and attractiveness for myogenesis cells. In conclusion, our findings suggest the D-MTJ ECM scaffold prepared in this study is a promising choice for muscle regeneration. STATEMENT OF SIGNIFICANCE: This study is the first to use decellularization technology obtaining the specifically decellularized myotendinous junction (D-MTJ) with well-preserved biphasic hierarchical structure and constituents, excellent mechanical properties and good biocompatibility. The D-MTJ was further proved to be efficient for muscle regeneration in vitro and in vivo, and the underlying mechanisms may be attributed to its specifically structure and constituents, improved myogenesis and good preservation of repair-related factors. Our study may provide basis for the decellularization of other biphasic hierarchical tissues and a platform for further studies on muscle fiber and tendon integrations in vitro.