Salicylate (SAL) has been recently implicated in the antidiabetic effect in humans. We assessed whether 5'-AMP-activated protein kinase (AMPK) in skeletal muscle is involved in the effect of SAL on glucose homeostasis. Rat fast-twitch epitrochlearis and slow-twitch soleus muscles were incubated in buffer containing SAL Intracellular concentrations of SAL increased rapidly (<5 min) in both skeletal muscles, and the Thr(172) phosphorylation of the a subunit of AMPK increased in a dose- and time-dependent manner. SAL increased both AMPK alpha 1 and AMPK alpha 2 activities. These increases in enzyme activity were accompanied by an increase in the activity of 3-O-methyl-D-glucose transport, and decreases in ATP, phosphocreatine, and glycogen contents. SAL did not change the phosphorylation of insulin receptor signaling including insulin receptor substrate 1, Akt, and p70 ribosomal protein S6 kinase. These results suggest that SAL may be transported into skeletal muscle and may stimulate AMPK and glucose transport via energy deprivation in multiple muscle types. Skeletal muscle AMPK might be part of the mechanism responsible for the metabolic improvement induced by SAL. (C) 2014 Elsevier Inc. All rights reserved.