Short-term exercise training in humans attenuates AMP-activated protein kinase (AMPK) activation during subsequent exercise conducted at the same absolute workload. Short-term 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) administration in rats mimics exercise training on skeletal muscle in terms of increasing insulin sensitivity, mitochondrial enzymes and GLUT-4 content but it is not known whether these adaptations are accompanied by reduced AMPK activation during subsequent exercise. We compared the effect of 10 days of treadmill training (60 min/day) with 10 days of AICAR administration (0.5 mg/g body weight i.p.) on subsequent AMPK activation during 45 min of treadmill exercise in male Sprague-Dawley rats. Compared with non-exercised control rats, acute exercise significantly (P<0.05) increased AMPK Thr¹⁷² phosphorylation (p-AMPK; 1.6-fold) and ACC Ser²¹⁸ phosphorylation (p-ACC; 4.9-fold) in the soleus and p-ACC 2.2-fold in the extensor digitorum longus (EDL). Ten days of exercise training abolished the increase in soleus p-AMPK and attenuated the increase in p-ACC (non-significant 2-fold increase). Ten days of AICAR administration also attenuated the exercise-induced increases in AMPK signaling in the soleus although not as effectively as 10 days of exercise training (non-significant 1.3-fold increase in p-AMPK; significant 3-fold increase in p-ACC). The increase in skeletal muscle 2-deoxyglucose uptake during exercise was greater after either 10 days of exercise training or AICAR administration. In conclusion, 10 days of AICAR administration substantially mimics the effect of 10 days training on attenuating skeletal muscle AMPK activation in response to subsequent exercise.