Aims/hypothesis: TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab GTPase-activating protein (RabGAP) that has been implicated in regulating GLUT4 trafficking. TBC1D1 can be phosphorylated by the AMP-activated protein kinase (AMPK) on Ser²³¹, which consequently interacts with 14-3-3 proteins. Given the key role for AMPK in regulating insulin-independent muscle glucose uptake, we hypothesised that TBC1D1-Ser²³¹ phosphorylation and/or 14-3-3 binding may mediate AMPK-governed glucose homeostasis. Methods: Whole-body glucose homeostasis and muscle glucose uptake were assayed in mice bearing a Tbc1d1^Ser231Ala-knockin mutation or harbouring skeletal muscle-specific Ampkα1/α2 (also known as Prkaa1/2) double-knockout mutations in response to an AMPK-activating agent, 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR). Exercise-induced muscle glucose uptake and exercise capacity were also determined in the Tbc1d1^Ser231Ala-knockin mice. Results: Skeletal muscle-specific deletion of Ampkα1/a2 in mice prevented AICAR-induced hypoglycaemia and muscle glucose uptake. The Tbc1d1^Ser231Ala-knockin mutation also attenuated the glucose-lowering effect of AICAR in mice. Glucose uptake and cell surface GLUT4 content were significantly lower in muscle isolated from the Tbc1d1^Ser231Ala-knockin mice upon stimulation with a submaximal dose of AICAR. However, this Tbc1d1^Ser231Ala-knockin mutation neither impaired exercise-induced muscle glucose uptake nor affected exercise capacity in mice. Conclusions/interpretation: TBC1D1-Ser²³¹ phosphorylation and/or 14-3-3 binding partially mediates AMPK-governed glucose homeostasis and muscle glucose uptake in a context-dependent manner.