While skeletal muscle has a high capacity for endogenous repair in acute injuries, volumetric muscle loss can leave long-lasting or permanent structural and functional deficits to the injured muscle and surrounding tissues. With clinical treatments failing to repair lost tissue, there is a great need for a tissue-engineered therapy to promote skeletal muscle regeneration. In this study, we aim to assess the potential for electrospun decellularized skeletal muscle extracellular matrix (dECM) with tunable physicochemical properties to control mouse myoblast growth and myotube formation. The material properties as well as cell behavior – growth and differentiation – were assessed in response to modulation of crosslinking and scaffold architecture. The fabrication of a bioactive dECM-based system with tunable physicochemical properties that can control myotube formation has several applications in skeletal muscle engineering and may bring the field one step closer to developing a therapy to address these unmet clinical needs. Electrospun decellularized skeletal muscle with tunable physicochemical properties controls myoblast growth and myotube formation. Electrospun decellularized skeletal muscle with tunable physicochemical properties controls myoblast growth and myotube formation.