AMP-activated protein kinase (AMPK) plays diverse roles and coordinates complex metabolic pathways for maintenance of energy homeostasis. This could be explained by the fact that AMPK exists as multiple heterotrimer complexes comprising a catalytic α subunit (α1, α2) and regulatory β (β1, β2) and (1, 2, 3) subunits, which are uniquely distributed across different cell types. There has been keen interest in developing specific and isoform-selective AMPK-activating drugs for therapeutic use and also as research tools. Moreover, establishing ways of enhancing cellular AMPK activity would be beneficial for both purposes. Here, we investigated if a recently-described potent AMPK-activator called 991, in combination with a commonly used activator AICAR or contraction, further enhances AMPK activity and glucose transport in mouse skeletal muscle ex vivo. Given that 3 is exclusively expressed in skeletal muscle and is implicated in contraction-induced glucose transport, we measured the activity of AMPK3- as well as ubiquitously expressed 1-containing complexes. We initially validated specificity of the antibodies for the assessment of isoform-specific AMPK activity using respective AMPK-deficient mouse models. We observed that a low dose of 991 (5 μM) stimulated a modest or negligible activity of both 1- and 3-containing AMPK complexes. Dual treatment with 991 and AICAR or 991 and contraction profoundly enhanced AMPK1-/3-complex activation and glucose transport compared to any of the single treatments. The study demonstrates a utility of dual activator approach to achieve a greater activation of AMPK and downstream physiological responses in various cell types including skeletal muscle.