The mitogen-activated protein kinase (MAPK)-activated protein kinases 2 and 3 (MK2 and MK3) are downstream targets of the p38 MAPK. Here, we demonstrate in a MK2/3 double knockout mouse model a shift to a slower oxidative phenotype in mixed fiber soleus muscle by performing histocytochemistry and gene expression analysis. The proportion of slow type I fibers was increased in MK2/3^-/- soleus muscle. Correspondingly, the expression of slow fiber type-specific genes encoding components of the contractile apparatus like myosin heavy and light chains was increased, whereas fast fiber-specific gene expression was decreased, as shown by mRNA microarray and on the level of protein. Likewise, genes involved in oxidative energy metabolism, including the master regulator of mitochondrial biogenesis, PPAR gamma coactivator-1α (PGC-1α), were upregulated. Furthermore, mitochondrial DNA expression was augmented. In line with these changes indicating an increase in oxidative capacity, the MK2/3^-/- soleus showed a higher resistance to fatigue compared with wild type soleus. Altered expression of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) isoforms of fast and slow type fibers, 1 and 2a, further indicated a shift towards increased slow fiber type proportion in MK2/3^-/- soleus. Gel mobility shift and transient transfection assays demonstrated that increased SERCA2 gene transcription in MK2/3^-/- soleus is partly mediated via a reduced MK2-dependent repressive function of the transcription factor Egr-1. Taken together, the data demonstrate that the p38 MAPK-MK2/3 axis affects fiber type composition and related function in mouse soleus muscle.