Reduction in the expression of sarcolipin (SLN), an inhibitor of sarco(endo)plasmic reticulum (SR) Ca²⁺-ATPase (SERCA), ameliorates severe muscular dystrophy in mice. However, the mechanism by which SLN inhibition improves muscle structure remains unclear. Here, we describe the previously unknown function of SLN in muscle differentiation in Duchenne muscular dystrophy (DMD). Overexpression of SLN in C₂C₁₂ resulted in decreased SERCA pump activity, reduced SR Ca²⁺ load, and increased intracellular Ca²⁺ () concentration. In addition, SLN overexpression resulted in altered expression of myogenic markers and poor myogenic differentiation. In dystrophin-deficient dog myoblasts and myotubes, SLN expression was significantly high and associated with defective cycling. The dystrophic dog myotubes were less branched and associated with decreased autophagy and increased expression of mitochondrial fusion and fission proteins. Reduction in SLN expression restored these changes and enhanced dystrophic dog myoblast fusion during differentiation. In summary, our data suggest that SLN upregulation is an intrinsic secondary change in dystrophin-deficient myoblasts and could account for the mishandling, which subsequently contributes to poor myogenic differentiation. Accordingly, reducing SLN expression can improve the cycling and differentiation of dystrophic myoblasts. These findings provide cellular-level supports for targeting SLN expression as a therapeutic strategy for DMD.