Regular endurance exercise induces skeletal muscle contractile and metabolic adaptations, conferring salutary health benefits, such as protection against the metabolic syndrome. The plasticity of skeletal muscle has been extensively investigated, but how the adaptive processes are precisely controlled is largely unknown. Using muscle-specific gene deletion in mice, we now show that p38γ mitogen-activated protein kinase (MAPK), but not p38α and p38β, is required for endurance exercise-induced mitochondrial biogenesis and angiogenesis, whereas none of the p38 isoforms are required for IIb-to-IIa fiber-type transformation. These phenotypic findings were further supported by microarray and real-time PCR analyses revealing contractile activity-dependent p38γ target genes, including peroxisome proliferator-activated receptor γ co-activator-1α (Pgc-1α) and vascular endothelial growth factor (Vegf), in skeletal muscle following motor nerve stimulation. Gene transfer-mediated overexpression of a dominant negative form of p38γ, but not that of p38α or p38β, blocked motor nerve stimulation-induced Pgc-1α transcription. These findings provide direct evidence for an obligated role of p38γ MAPK-PGC-1α regulatory axis in endurance exercise-induced metabolic adaptation, but not contractile adaptation, in skeletal muscle.