Peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) promotes mitochondrial biogenesis and slow fiber formation in skeletal muscle. We hypothesized that activation of the p38 mitogen-activated protein kinase (MAPK) pathway in response to increased muscle activity stimulated Pgc-1alpha gene transcription as part of the mechanisms for skeletal muscle adaptation. Here we report that a single bout of voluntary running induced a transient increase of Pgc-1alpha mRNA expression in mouse plantaris muscle, concurrent with an activation of the p38 MAPK pathway. Activation of the p38 MAPK pathway in cultured C2C12 myocytes stimulated Pgc-1alpha promoter activity, which could be blocked by the specific inhibitors of p38, SB203580 and SB202190, or a dominant negative p38. Furthermore, the p38-mediated increase in Pgc-1alpha promoter activity was enhanced by increased expression of the downstream transcription factor ATF2 and completely blocked by ATF2DeltaN, a dominant negative ATF2. Skeletal muscle-specific expression of a constitutively active activator of p38, MKK6E, in transgenic mice resulted in enhanced Pgc-1alpha and cytochrome oxidase IV protein expression in fast-twitch skeletal muscles. These findings suggest that contractile activity-induced activation of the p38 MAPK pathway promotes Pgc-1alpha gene expression and skeletal muscle adaptation.