AbstractThe reconstruction of skeletal muscle tissue is currently performed by transplanting a muscle tissue graft from local or distant sites of the patient's body, but this practice leads to donor site morbidity in case of large defects. With the aim of providing an alternative treatment approach, skeletal muscle tissue formation potential of human myoblasts and human menstrual blood derived mesenchymal stem cells (hMB‐MSCs) on synthetic [poly(l‐lactide‐co‐caprolactone), 70:30] scaffolds with oriented microfibers, human muscle extracellular matrix (ECM), and their hybrids was investigated in this study. The reactive muscle ECM pieces were chemically crosslinked to the synthetic scaffolds to produce the hybrids. Cell proliferation assay WST‐1, scanning electron microscopy (SEM), and immunostaining were carried out after culturing the cells on the scaffolds. The ECM and the synthetic scaffolds were effective in promoting spontaneous myotube formation from human myoblasts. Anisotropic muscle patch formation was more successful when human myoblasts were grown on the synthetic scaffolds. Nonetheless, spontaneous differentiation could not be induced in hMB‐MSCs on any type of the scaffolds. Human myoblast‐synthetic scaffold combination is promising as a skeletal muscle patch, and can be improved further to serve as a fast integrating functional patch by introducing vascular and neuronal networks to the structure. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 879–890, 2017.