5′AMP‐activated protein kinase (AMPK) is a key regulator of cellular metabolism and is regulated in muscle during exercise. We have previously established that only three of 12 possible AMPK α/β/γ‐heterotrimers are present in human skeletal muscle. Previous studies describe discrepancies between total AMPK activity and regulation of its target acetyl‐CoA‐carboxylase (ACC)β. Also, exercise training decreases expression of the regulatory γ3 AMPK subunit and attenuates α2 AMPK activity during exercise. We hypothesize that these observations reflect a differential regulation of the AMPK heterotrimers. We provide evidence here that only the α2/β2/γ3 subunit is phosphorylated and activated during high‐intensity exercise in vivo. The activity associated with the remaining two AMPK heterotrimers, α1/β2/γ1 and α2/β2/γ1, is either unchanged (20 min, 80% maximal oxygen uptake ) or decreased (30 or 120 s sprint‐exercise). The differential activity of the heterotrimers leads to a total α‐AMPK activity, that is decreased (30 s trial), unchanged (120 s trial) and increased (20 min trial). AMPK activity associated with the α2/β2/γ3 heterotrimer was strongly correlated to γ3‐associated α‐Thr‐172 AMPK phosphorylation (r2= 0.84, P < 0.001) and to ACCβ Ser‐221 phosphorylation (r2= 0.65, P < 0.001). These data single out the α2/β2/γ3 heterotrimer as an important actor in exercise‐regulated AMPK signalling in human skeletal muscle, probably mediating phosphorylation of ACCβ.