Skeletal muscle has a remarkable capacity of regeneration after injury, but the cellular behavior and the regulatory network coordinating different steps of this repair process remain elusive. RNA-binding proteins play key roles in the post-transcriptional regulation of gene expression and are implicated in the maintenance of tissue homeostasis and plasticity. Rbm24 is required for myogenic differentiation during early development, but its function in adult muscle is open for investigation. Here we show that it exerts dynamic functions during muscle regeneration in mice. Consistent with its dynamic subcellular localization during embryonic muscle development, Rbm24 also displays cytoplasm to nucleus translocation during the differentiation of C2C12 myoblasts. In adult mice, Rbm24 mRNA is highly expressed in slow-twitch muscles, and Rbm24 protein is restricted to the myonucleus of myofibers. Upon injury, Rbm24 protein is upregulated in regenerating myofibers and rapidly accumulates in the myonucleus of nascent myofibers. By using satellite cell transplantation, we find that Rbm24 functions sequentially to regulate the differentiation of myofibers and the regeneration of damaged tissues. It is required for myogenin mRNA expression at early stages of muscle injury and for muscle-specific pre-mRNA alternative splicing at late stages of regeneration. These results identify Rbm24 as a multifaceted regulator of myoblast differentiation and function. They also provide insights into the molecular pathway orchestrating the expression of myogenic factors and muscle functional proteins during regeneration.