5′ AMP-activated protein kinase (AMPK) activation may be part of the exercise-induced process that enhances insulin sensitivity. Independent of exercise, acute prior treatment of skeletal muscles isolated from young rats with a pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), causes subsequently improved insulin-stimulated glucose uptake (GU). However, efficacy of a single prior AICAR exposure on insulin-stimulated GU in muscles from old animals has not been studied. The purpose of this study was to determine whether brief, prior exposure to AICAR (3.5 hours before GU assessment) leads to subsequently increased GU in insulin-stimulated skeletal muscles from old rats. Epitrochlearis muscles from 24 month-old male rats were isolated and initially incubated ±AICAR (60 minutes), followed by incubation without AICAR (3 hours), then incubation ±insulin (50 minutes). Muscles were assessed for GU (via 3-O-methyl-[3H]-glucose accumulation) and site-specific phosphorylation of key proteins involved in enhanced GU, including AMPK, Akt, and Akt substrate of 160 kDa (AS160), via western blotting. Prior ex vivo AICAR treatment resulted in greater GU by insulin-stimulated muscles from 24 month-old rats. Prior AICAR treatment also resulted in greater phosphorylation of AMPK (T172) and AS160 (S588, T642 and S704). GLUT4 protein abundance was unaffected by prior AICAR and/or insulin treatment. These findings demonstrate that skeletal muscles from older rats are susceptible to enhanced insulin-stimulated GU after brief activation of AMPK by prior AICAR. Consistent with earlier research using muscles from young rodents, increased phosphorylation of AS160 is implicated in this effect that was not attributable to altered GLUT4 glucose transporter protein abundance.