Clinical trials have shown that direct activators of an evolutionary-conserved metabolic sensor, AMP-activated protein kinase (AMPK), are beneficial in preventing/treating a range of metabolic disorders, including type 2 diabetes. These activators, including 991/MK-8722, bind at regulatory site, termed the allosteric drug and metabolite (ADaM-site) at the interface between the catalytic alpha subunit and regulatory beta subunit. In addition, another approach for direct activation of AMPK is through mimicking its natural ligands, AMP/ADP, by binding to the nucleotide site in the y3 subunit, e.g. 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Interestingly, their ability to influence glucose homeostasis is reported to be through activation of AMPK complexes found within the skeletal muscle. The AMPKy3 is unique as an AMPK subunit isoform since it is selectively expressed in glycolytic skeletal muscle fibres. To gain more insight into the y3 isoform-specific regulation of glucose metabolism, we used a y3 knock-out (y3-/-) mouse model to investigate the effects of the AICAR and 991/MK-8722. We confirm that y3 isoform expression and activity is high glycolytic muscles, including extensor digitorum longus (EDL), whereas there is very little detected in oxidative soleus (SOL) muscle. We show that AICAR and 991/MK-8722 stimulated glucose uptake in both EDL and SOL muscles. Interestingly, only the ability of AICAR to induce glucose uptake was blunted in EDL muscle taken from y3-/- mice. Consistent with this, whole-body glucose clearance was also impaired in y3-/- mice in response to an AICAR, whilst MK-8722 lowered blood glucose similarly between WT and y3-/- mice. Despite the presence of y1-containing complexes, which are the most predominant complexes in both EDL and SOL muscles, this suggests that different AMPKy isoforms play different roles in glucose homeostasis in response to AICAR. We further looked at de novo glycogen synthesis and glucose utilisation in skeletal muscle. Despite similar basal glucose uptake between muscles from WT and y3-/-, y3-deficiency results in lower basal glycogen content only in glycolytic muscles. We show that this is not due to an inability to synthesise glycogen de novo, since MK-8722 was able to increase glycogen levels in EDL. Instead, we show that the decrease in basal glycogen in y3-/- EDL, may be linked lower expression of UDP-glucose pyrophosphorylase 2 (UGP2). This suggests that AMPKy3 may play a role in steering the molecular fate of glucose inside the cell. Taken together, whilst we show that AMPKy3 is dispensable for ex vivo glucose transport and in vivo glucose homeostasis in response to ADaM-site AMPK activators, nucleotide-mediated activation of y3 by AICAR is impaired. Further research is required to understand the nucleotide regulation of different y-containing AMPK complexes. This differential activation mechanism could be exploited to specifically target AMPKy3 complexes in the muscle, and potentially avoid deleterious effects of activation of AMPK complexes in other tissues.