Skeletal muscle is a major tissue for storing glucose as glycogen, converting glucose to lactate and fully oxidising glucose to CO2. Muscle also has a limited capacity for gluconeogenesis converting lactate and alanine to glycogen. This gluconeogenesis requires FBP2, a muscle specific form of fructose bisphosphatase that converts fructose-1,6-bisphosphate (F-1,6-bisP) to fructose-6-phosphate (F-6-P) opposing the activity of the ATP-consuming enzyme phosphofructokinase (PFK). In mammalian muscle, the activity of FBP2 is normally 100 times lower than PFK and therefore energy wasting between F-1,6-bisP/F-6-P cycling is low, although, cycling can increase with an increase in muscle metabolism during exercise, cold exposure or thyroid treatment. In an attempt to increase substrate cycling between F-6-P and F-1,6-bisP and alter glucose metabolism, we overexpressed FBP2 using a muscle-specific adeno-associated virus (AAV-tMCK-FBP2). AAV was injected into the right tibialis muscle of 60g male Wistar rats, while the left tibialis received a saline injection. The animals were then fed a chow or 45% fat diet (HFD) for 5 weeks after which euglycaemic hyperinsulinaemic clamps was performed. The glucose infusion rate was significantly lower in HFD compared to chow (21.2±2.3 vs 37.1±2.8 mg/kg/min, p<0.001) indicating whole body insulin resistance. Transfection of the right tibialis with AAV-tMCK-FBP2 increased FBP2 activity 10 fold on average in chow and HFD rats (p<0.0001). Insulin-stimulated glucose uptake was significantly lower in tibialis of HFD compared to chow rats (2-way ANOVA, diet p=0.01). Overexpression of FBP2 significantly increased insulin-stimulated glucose uptake in tibialis of chow animals (control 14.3±1.7; FBP2 17.6±1.6µmol/min/100g) and HFD animals (control 9.6±1.1; FBP2 11.2±1.1 µmol/min/100g) (2-way ANOVA, FBP2 p<0.001). These results suggest that overexpressing FBP2 can increase the capacity for cycling between F-6-P and F-1,6-P, which can increase the metabolism of glucose by introducing a futile cycle in muscle.