Insulin plays a critical role in regulating cellular metabolism by promoting cellular glucose and fatty acid (FA) uptake, lipid synthesis and inhibiting gluconeogenesis and lipolysis. Although mechanisms of FA uptake have been described in myocytes, FA uptake in adipocytes remains poorly characterised. AMP-activated protein kinase (AMPK) has been proposed to be a potential therapeutic target for obesity and type 2 diabetes due to its critical role in the maintenance of cellular and whole-body energy balance. AMPK activation by AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide) is associated with an increase in GLUT4 translocation and glucose uptake in skeletal muscle. However, AICAR has been previously demonstrated to increase basal, yet inhibit insulin-stimulated glucose uptake. AMPK activation has been reported to increase FA uptake in myocytes in a manner analogous to glucose uptake. This study aimed to characterise the effect of AMPK activity on FA uptake and the expression of FA transporter proteins. The effect of AMPK activators on glucose or FA uptake was assessed in 3T3-L1 adipocytes incubated with AICAR (2 mM, 60 min) and/or the direct AMPK activator A769662 (300 µM, 60 min) prior to stimulation with insulin. 2-[3H]-deoxy-D-glucose or [3H]palmitate uptake was subsequently assessed. To examine whether lack of AMPK activity influenced expression of glucose and FA transporters, the levels of glucose transporter (SLC2A1 and SLC2A4) and FA transporter (SLC27A1, SLC27A4 and CD36) mRNA were assessed by RT-PCR in epidydimal adipose tissue from wild-type and AMPKα1 knockout mice fed chow or high fat diet. Insulin significantly increased glucose uptake ~2.5-fold (mean ± SEM, n=5, p<0.0001, 2-way ANOVA), an effect that was significantly inhibited by preincubation with A769662 (~70%, n=5, p<0.0001, 2-way ANOVA) and tended towards inhibition with the less-selective AMPK activator AICAR (~30%, n=5, p=0.13, 2-way ANOVA). Recent studies have indicated that insulin-stimulated FA uptake is mediated by mTOR, yet inhibition of mTOR with rapamycin had no effect on insulin-stimulated glucose uptake. Insulin stimulation significantly increased [3H]palmitate uptake ~2-fold in 3T3-L1 adipocytes, an effect inhibited by A769662 (n=4, p<0.01, 2-way ANOVA). There were no significant differences in the levels of mRNA encoding FA and glucose transporters in adipose tissues between wild type and AMPKα1 knockout mice or between diets. These data indicate that AMPK activation inhibits insulin-stimulated FA uptake in 3T3-L1 adipocytes, analogous to the inhibitory effect of AMPK on insulin-stimulated glucose uptake. Furthermore, lack of AMPKα1 has no effect on levels of glucose or FA transporter mRNA in adipose tissue. These data highlight the contrasting actions of AMPK activation on nutrient influx in adipocytes compared with myocytes, where AMPK activation has been reported to increase both glucose and FA uptake under basal and insulin-stimulated conditions.