Caffeine is the most widely behaviorally active substance consumed in the world. We have recently showed that chronic caffeine intake prevents the development of insulin resistance (IR) in diet-induced IR rats (Conde et al. 2012). In contrast, acute caffeine administration decreases insulin sensitivity in a dose dependent manner (Sacramento et al. 2012). However, the mechanism behind the distinct effects of acute and chronic caffeine administration on insulin sensitivity is not clarified. Herein we have investigated if the mechanism that contributes to the differences between acute and chronic caffeine effects on insulin sensitivity is mediated by altered insulin/AMPK signaling pathway in skeletal muscle. Experiments were performed in 3 months Wistar rats anaesthetized with pentobarbitone (60mg/Kg, ip). Six groups of animals were used to evaluate if the effects of chronic caffeine intake on insulin sensitivity are mediated by altered insulin/AMPK pathway. A control group (fed with a sham diet), an high sucrose (HSu; 35% sucrose in drinking water, 28 days) and an high fat (HF; lipid rich diet with 45% fat, 21days) groups were randomly divided into two subgroups, treated and not treated with 1g/l caffeine during 15 days. Insulin sensitivity was measured by an insulin tolerance test (ITT). Acute caffeine administration effects on insulin/AMPK signaling pathway were evaluated in animals submitted to an i.v. administration of different doses of caffeine (0.001-5 µM) 15 minutes before the ITT. Blood was collected by heart puncture and skeletal muscle was removed for Western-blot detection and quantification of insulin receptor, insulin receptor phosphor-Tyr1322, AMPKα1, AMPKα1 Thr172 and Glut4 transporters. Glut4 expression decreased by 59.35% in HSu animals, an effect not modified by chronic caffeine. In contrast, acute caffeine administration decreased significantly Glut4 expression at 0.5μM of caffeine (p<0.01). HSu and HF diets decreased significantly AMPKα1 expression by 70.45% and 33.93%, respectively. Chronic caffeine intake increased significantly AMPKα1 in HSu animals (42.54%) and in HF group AMPK α1 expression was restored to control levels. AMPK α1 activity decreased significantly in HF animals (44.74%), however chronic caffeine intake did modify those values. In opposite, acute caffeine intake did not alter AMPK α1 expression. Insulin receptor decreased significantly in HF animals (59.43%) but chronic caffeine intake did not alter this value. Acute and chronic caffeine did not altered insulin receptor phosphor-Tyr1322 expression. In conclusion, the effect of chronic caffeine intake on insulin sensitivity is not mediated by altered insulin and/or AMPK signaling pathway. In contrast, the effect of acute caffeine administration on insulin sensitivity seems to involve a decrease in Glut4 transporters.