Long non-coding RNAs (lncRNAs) regulate a series of physiological processes and play an important role in development, metabolism and disease. Our previous studies showed that lncRNAs involved in skeletal muscle differentiation. Here, we demonstrated that lncRNA Has2os is highly expressed in skeletal muscle and significantly elevated during skeletal cell differentiation. The knockdown of Has2os inhibited myocyte fusion and impeded the expression of the myogenic factors MyHC and Mef2C. Mechanically, Has2os regulates skeletal muscle differentiation by inhibiting the JNK/MAPK signaling pathway. Furthermore, we also revealed that Has2os is involved in the early stage of regeneration after muscle injury, and the JNK/MAPK signaling pathway is activated at both protein and mRNA levels during early repair. Our results demonstrate the new function of lncRNA Has2os, which plays crucial roles during skeletal muscle differentiation and muscle regeneration, providing a basis for the therapy of lncRNA-related muscle diseases.