AMP-activated protein kinases (AMPK) are heterotrimeric, αβγ, serine/threonine kinases. The γ3-AMPK subunit is particularly interesting in muscle physiology because 1) it is specifically expressed in skeletal muscle, 2) α2β2γ3 is the AMPK heterotrimer activated during exercise in humans, and 3) it is down-regulated in humans after a training period. However, mechanisms underlying this decrease of γ3-AMPK expression remained unknown. We investigated whether the expression of AMPK subunits and particularly that of γ3-AMPK are regulated by the PPARβ pathway. We report that PPARβ activation with GW0742 induces a rapid (2 h) and sustained down-regulation of γ3-AMPK expression both in mouse skeletal muscles and in culture myotubes. Concomitantly, phosphorylation levels of both AMPK and acetyl-coenzyme A carboxylase are rapidly modified. The γ3-AMPK down-regulation is also observed in muscles from young and adult transgenic mice with muscle-specific overexpression of peroxisome proliferator-activated receptor β (PPARβ). We showed that γ3-AMPK down-regulation is a rapid physiological muscle response observed in mouse after running exercise or fasting, two situations leading to PPARβ activation. Finally, using C2C12, we demonstrated that dose and time-dependent down-regulation of γ3-AMPK expression upon GW0742 treatment, is due to decrease γ3-AMPK promoter activity.