We tested the hypothesis that repeated activation of AMPK induces mitochondrial and glucose membrane transporter gene/protein expression via a peroxisome proliferator activated receptor Upsilon co-activator (PGC)-1alpha dependent mechanism. Whole body PGC-1alpha knockout (KO) and littermate wild type (WT) mice were given either a single or repeated subcutaneous injection of the AMPK activator AICAR or saline. Skeletal muscles were dissected either 1h or 4h after the single AICAR treatment or 24h after the last injection following the repeated AICAR treatment. Repeated AICAR treatment increased GLUT4, cytochrome c oxidase (COX)I and cytochrome (cyt) c protein expression ~10-40% relative to saline in white muscles of the WT mice, but not of the PGC-1alpha KO mice. In line, GLUT4 and cyt c mRNA content increased 30-60% 4h after a single AICAR injection relative to saline only in WT mice. One hour after a single AICAR treatment, phosphorylation of AMP-activated protein kinase (AMPK) and the downstream target acetyl-coenzyme A carboxylase (ACC) increased in all muscles investigated independent of genotype, indicating a normal AICAR induced AMPK signalling in the absences of PGC-1alpha. Also, the hexokinase (HK)II expression response was similar in muscles of WT and PGC-1alpha mice both after single and repeated AICAR treatments. This confirms that the HKII gene is regulated independently of PGC-1alpha and importantly that the effect of AICAR was maintained with repeated AICAR treatments in both WT and PGC-1alpha KO mice. In conclusion, we here provide genetic evidence for a major role of PGC-1alpha in AMPK mediated regulation of mitochondrial and glucose membrane transport protein expression in skeletal muscle.