FoxO1 is a transcription factor thought to link transcription to metabolic control¹, yet little is known regarding the activation state of FoxO1 on muscle glucose metabolism. We studied the effect of a high-fat diet (HFD; 42% energy from fat) for 16wk on whole-body and tissue-specific glucose homeostasis in male C57BL/6 mice expressing a muscle-specific, constitutively active (CA)-FoxO1 (foxo1^Tg), or wild-type (WT) littermates. After 4wk of the HFD, foxo1^Tg mice had lower body weight due to reduced lean mass, although as a percentage of body weight lean mass (72±2 vs. 73±2% for WT) and fat mass (25±2% for foxo1^Tg and WT) were similar. Glucose tolerance at 4wk, determined by the glucose area under the curve (AUC) during a glucose tolerance test (GTT), was improved in foxo1^Tg mice (1601±75 vs. 1969±122mM/min for WT, p<0.02). The glucose infusion rate required to maintain euglycaemia (GIR) during a hyperinsulinaemic-euglycaemic clamp was increased ~40% in foxo1^Tg mice, and muscle glucose uptake tended to be elevated (48±11 vs. 27±4µmol/100g/min for WT, p=0.08). However, the improved glucose tolerance/insulin sensitivity seen at 4wk of the HFD in foxo1^Tg mice was not present after 16wk. Indeed, AUC during a GTT was similar to WT, GIR was only elevated ~15% in foxo1^Tg mice, while insulin-stimulated muscle glucose uptake was unaltered (12±2 vs. 14±1µmol/100g/min for WT). foxo1^Tg mice had reduced lean mass (55±2 vs. 63±1% for WT, p<0.05), and had gained more fat mass (43±1 vs. 35±1% for WT, p<0.01). Thus, during the early stages of a HFD, a CA-FoxO1 is associated with improved whole-body and tissue-specific glucose homeostasis. This beneficial effect is lost after a prolonged period on HFD, and associated with enhanced fat mass and reduced lean mass, which was expected since FoxO1 can transcriptionally activate the muscle specific E3 ligases Murf1² and MAFbx³.