Skeletal muscle formation and regeneration require myoblast fusion to form multinucleated myotubes or myofibers, yet their molecular regulation remains incompletely understood. We show here that the levels of extra- and/or pericellular chondroitin sulfate (CS) chains in differentiating C2C12 myoblast culture are dramatically diminished at the stage of extensive syncytial myotube formation. Forced down-regulation of CS, but not of hyaluronan, levels enhanced myogenic differentiation in vitro. This characteristic CS reduction seems to occur through a cell-autonomous mechanism that involves HYAL1, a known catabolic enzyme for hyaluronan and CS. In vivo injection of a bacterial CS-degrading enzyme boosted myofiber regeneration in a mouse cardiotoxin-induced injury model and ameliorated dystrophic pathology in mdx muscles. Our data suggest that the control of CS abundance is a promising new therapeutic approach for the treatment of skeletal muscle injury and progressive muscular dystrophies.