Type 2 diabetes mellitus (T2DM) is a serious metabolic disease in need of new treatments, and AMP-activated protein kinase (AMPK) is a strong potential therapeutic target due to its integral role in glucose homeostasis. AMPK exists as an αβγ heterotrimer and multiple isoforms exist for each subunit (α1/2, β1/2, γ1/2/3) allowing the formation of 12 unique AMPK complexes in a 1:1:1 ratio. Among these, the α2β2γ3 complex emerges as an ideal target for novel T2DM treatments due to its muscle-restricted tissue expression profile, a tissue heavily implicated in appropriate glucose handling. However, the lack of structural and functional information on this isoform has hindered drug development. This study aimed to characterize the function and regulatory role of a unique, 168 residue NH2-terminal extension (NTE) in the AMPKγ3 subunit. Utilising HEK293T cells we were able to express and purify AMPK complexes devoid of the entire γ3-NTE, this resulted in a significant increase in basal activity without affecting small molecule allosteric AMPK activation. Using complementary biophysical techniques including hydrogen-deuterium exchange-mass spectrometry, surface plasmon resonance, zero-distance chemical crosslinking and co-pulldowns, a specific region from the γ3-NTE, predicted to have a helical structure, was found to directly interact with the α2 kinase domain (α2-KD). AlphaFold3 was utilized to structurally visualize this γ3-NTE helix bound to the α2-KD of the α2β2γ3 complex, importantly the predicted model corroborates with all biochemical data. Recent results point to a regulatory mechanism for modulating the extent of inhibition from the γ3-NTE, indicating the γ3-NTE/α2-KD interaction could be therapeutically exploited. These findings have instigated the groundwork for the development of novel T2DM therapies targeting AMPK activation in skeletal muscle.