The aim was to test if the transcriptional coactivator peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)1alpha regulates the content of pyruvate dehydrogenase (PDH)-E1alpha and influences PDH activity through regulation of PDK4 expression and subsequently PDH phosphorylation. PGC-1alpha whole body knockout (KO), muscle specific PGC-1alpha overexpressing mice (MCK PGC-1alpha) and littermate wild type (WT) mice underwent two interventions known to affect PDH. Quadriceps muscles were removed from fed and 24h fasted mice as well as at 6h of recovery after 1h running and from mice that did not run acutely. PDH-E1alpha protein content and PDH-E1alpha phosphorylation were lower in PGC-1alpha KO and higher in MCK PGC-1alpha mice at rest, but while MCK PGC-1alpha had higher PDK4 protein content, KO of PGC-1alpha had no effect on PDK4 protein content. The differences in phosphorylation partly vanished when expressing phosphorylation relative to the PDH-E1alpha content with only a maintained elevated phosphorylation in MCK PGC-1alpha mice. Fasting up-regulated PDK4 protein in both PGC-1alpha KO, MCK PGC-1alpha and WT mice, but this was not consistently associated with increased PDH-E1alpha phosphorylation. Down-regulation of PDHa activity at 6h recovery from exercise in both the PGC-1alpha KO and MCK PGC-1alpha and the association between PDH-E1alpha phosphorylation and PDHa activity in PGC-1alpha KO mice indicate that PGC-1alpha is not required for these responses. In conclusion, PGC-1alpha regulates PDH-E1alpha protein content parallel with mitochondrial oxidative proteins, but does not seem to influence PDH regulation in mouse skeletal muscle in response to fasting and in recovery from exercise.