Duchenne muscular dystrophy (DMD) is characterized by progressive muscle wasting following repeated muscle damage and inadequate regeneration. Impaired myogenesis and differentiation play a major role in DMD as well as intracellular calcium (Ca) mishandling. Ca release from the sarcoplasmic reticulum is mostly mediated by the type 1 ryanodine receptor (RYR1) that is required for skeletal muscle differentiation in animals. The study objective was to determine whether altered RYR1-mediated Ca release contributes to myogenic differentiation impairment in DMD patients. The comparison of primary cultured myoblasts from six boys with DMD and five healthy controls highlighted delayed myoblast differentiation in DMD. Silencing expression using specific si-RNA in a healthy control induced a similar delayed differentiation. In DMD myotubes, resting intracellular Ca concentration was increased, but RYR1-mediated Ca release was not changed compared with control myotubes. Incubation with the RYR-calstabin interaction stabilizer S107 decreased resting Ca concentration in DMD myotubes to control values and improved calstabin1 binding to the RYR1 complex. S107 also improved myogenic differentiation in DMD. Furthermore, intracellular Ca concentration was correlated with endomysial fibrosis, which is the only myopathologic parameter associated with poor motor outcome in patients with DMD. This suggested a potential relationship between RYR1 dysfunction and motor impairment. Our study highlights RYR1-mediated Ca leakage in human DMD myotubes and its key role in myogenic differentiation impairment. RYR1 stabilization may be an interesting adjunctive therapeutic strategy in DMD.