TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) is a Rab-GAP (GTPase-activating protein) that is highly expressed in skeletal muscle, but little is known about TBC1D1 regulation and function. We studied TBC1D1 phosphorylation on three predicted AMPK (AMP-activated protein kinase) phosphorylation sites (Ser²³¹, Ser⁶⁶⁰ and Ser⁷⁰⁰) and one predicted Akt phosphorylation site (Thr⁵⁹⁰) in control mice, AMPKα2 inactive transgenic mice (AMPKα2i TG) and Akt2-knockout mice (Akt2 KO). Muscle contraction significantly increased TBC1D1 phosphorylation on Ser²³¹ and Ser⁶⁶⁰, tended to increase Ser⁷⁰⁰ phosphorylation, but had no effect on Thr⁵⁹⁰. AICAR (5-aminoimidazole-4-carboxyamide ribonucleoside) also increased phosphorylation on Ser²³¹, Ser⁶⁶⁰ and Ser⁷⁰⁰, but not Thr⁵⁹⁰, whereas insulin only increased Thr⁵⁹⁰ phosphorylation. Basal and contraction-stimulated TBC1D1 Ser²³¹, Ser⁶⁶⁰ and Ser⁷⁰⁰ phosphorylation were greatly reduced in AMPKα2i TG mice, although contraction still elicited a small increase in phosphorylation. Akt2 KO mice had blunted insulin-stimulated TBC1D1 Thr⁵⁹⁰ phosphorylation. Contraction-stimulated TBC1D1 Ser²³¹ and Ser⁶⁶⁰ phosphorylation were normal in high-fat-fed mice. Glucose uptake in vivo was significantly decreased in tibialis anterior muscles overexpressing TBC1D1 mutated on four predicted AMPK phosphorylation sites. In conclusion, contraction causes site-specific phosphorylation of TBC1D1 in skeletal muscle, and TBC1D1 phosphorylation on AMPK sites regulates contraction-stimulated glucose uptake. AMPK and Akt regulate TBC1D1 phosphorylation, but there must be additional upstream kinases that mediate TBC1D1 phosphorylation in skeletal muscle.