Skeletal myogenesis is associated with the activation of four muscle regulatory factors (MRFs): Myf5, MyoD, Myogenin and MRF4. Here we report that p38 mitogen-activated protein kinase represses the transcriptional activity of MRF4 (involved in late stages of myogenesis), resulting in downregulation of specific muscle genes. MRF4 is phosphorylated in vitro and in vivo by p38 on two serines (Ser31 and Ser42) located in the N-terminal transactivation domain, resulting in reduced MRF4-mediated transcriptional activity. In contrast, nonphosphorylatable MRF4 mutants display increased transcriptional activity and are able to advance both myoblast fusion and differentiation. We also show that expression of desmin and alpha-actin, but not muscle creatin kinase, decreased at late stages of muscle differentiation, correlating with the induction of MRF4 and p38 activation. Accordingly, inhibition of p38 during late myogenesis results in the upregulation of both desmin and alpha-actin. We propose that repression of MRF4 activity by p38 phosphorylation may represent a new mechanism for the silencing of specific muscle genes at the terminal stages of muscle differentiation.