Limb-girdle muscular dystrophy type 2L (LGMD2L) is a myopathy arising from mutations in ANO5; however, information about the contribution of ANO5 to muscle physiology is lacking. To explain the role of ANO5 in LGMD2L, we previously hypothesized that ANO5-mediated phospholipid scrambling facilitates cell–cell fusion of mononucleated muscle progenitor cells (MPCs), which is required for muscle repair. Here, we show that heterologous overexpression of ANO5 confers Ca²⁺-dependent phospholipid scrambling to HEK-293 cells and that scrambling is associated with the simultaneous development of a nonselective ionic current. MPCs isolated from adult Ano5^−/− mice exhibit defective cell fusion in culture and produce muscle fibers with significantly fewer nuclei compared with controls. This defective fusion is associated with a decrease of Ca²⁺-dependent phosphatidylserine exposure on the surface of Ano5^−/− MPCs and a decrease in the amplitude of Ca²⁺-dependent outwardly rectifying ionic currents. Viral introduction of ANO5 in Ano5^−/− MPCs restores MPC fusion competence, ANO5-dependent phospholipid scrambling, and Ca²⁺-dependent outwardly rectifying ionic currents. ANO5-rescued MPCs produce myotubes having numbers of nuclei similar to wild-type controls. These data suggest that ANO5-mediated phospholipid scrambling or ionic currents play an important role in muscle repair.