Skeletal muscle differentiation entails organized sequential events, including cell cycle arrest of proliferating myoblast cells and cell fusion, which lead to the formation of multinucleated myotubes. This process involves both transcriptional and post-transcriptional regulation of the gene expression of myogenic proteins, as well as cell-cycle related proteins. RNA-binding proteins bind to specific sequences of target RNA and regulate gene expression in a post-transcriptional manner. However, few tissue-specific RNA binding proteins have been identified. Herein, we report that the RNA binding proteins Rbm24 and Rbm38 were found to be preferentially expressed in muscle during differentiation in vitro. Further, knockdown of either by RNA interference suppressed cell-cycle arrest and delayed myogenic differentiation in C2C12 cells. In contrast, over-expression of Rbm24 or Rbm38 induced cell cycle arrest, and then had a positive effect on myogenic differentiation. Immunoprecipitation-RT-PCR analysis using tagged Rbm proteins indicated that Rbm38 binds to the p21 transcript in vivo. Consistent with this, differentiation of Rbm38 knockdown cells was rescued by over-expression of p21. Together, our results suggest that Rbm38 plays a crucial role in cell cycle arrest and myogenic differentiation via its binding to p21.