5′‐AMP‐activated protein kinase (AMPK) activity is increased during exercise in an intensity‐ and glycogen‐dependent manner. We previously reported that a mutation in the AMPKγ3 subunit (Prkag3^225Q) increases AMPK activity and skeletal muscle glycogen content. Transfection experiments revealed the R225Q mutation is associated with high basal AMPK activity and diminished AMP dependence. Thus, the R225Q mutation can be considered a loss‐of‐function mutation that abolished allosteric regulation by AMP/ATP, causing increased basal AMPK activity. We used AMPKγ3 transgenic (Tg‐Prkag3^225Q) and knockout (Prkag3^−/−) mice to determine the relationship between AMPK activity, glycogen content, and ergogenics (ability to perform work) in isolated extensor digitorum longus skeletal muscle after contractions induced by electrical stimulation. Contraction‐induced AMPK activity was inversely coupled to glycogen content in wild‐type and Tg‐Prkag3^225Q mice, but not in Prkag3^−/− mice, highlighting a partial feedback control of glycogen on contraction‐induced AMPK activity in the presence of a functional AMPKγ3 isoform. Skeletal muscle glycogen content was positively correlated to work performance, regardless of genotype. Thus, chronic activation of AMPK by the Prkag3^225Q mutation directly influences skeletal muscle ergogenics by enhancing glycogen content. In conclusion, functional studies of the AMPKγ3 isoform further support the close connection between glycogen content and exercise performance in skeletal muscle.—Barnes, B. R., Glund, S., Long, Y. C., Hjälm, G., Andersson, L., Zierath, J. R. 5'‐AMP‐activated protein kinase regulates skeletal muscle glycogen content and ergogenics. FASEB J. 19, 773–779 (2005)