We evaluated the effect of skeletal muscle mitochondrial uncoupling on energy and glucose metabolism under different dietary challenges. Transgenic HSA-mUCP1 mice with ectopic expression of uncoupling protein 1 in skeletal muscle and wildtype littermates were fed for 3 months semi-synthetic diets with varying macronutrient ratios (energy % carbohydrate:protein:fat): HCLF: (41:42:17), HCHF (41:16:43); LCHF (11:45:44). Body composition, energy metabolism and insulin resistance were assessed by quantitative NMR, indirect calorimetry, and insulin tolerance test, respectively. Finally, plasma metabolites were measured and gene expression of key proteins was determined in muscle, liver and white fat using real time PCR. In wildtype, both high fat diets led to an increase in body weight and fat. HSA-mUCP1 mice considerably increased body fat on the HCHF diet but stayed lean on the other diets. Irrespective of differences in body fat content, HSA-mUCP1 mice were much more insulin sensitive and had lower insulin levels and liver triglycerides compared to wildtype. Respiratory quotient and gene expression indicated overall higher carbohydrate oxidation of HSA-mUCP1 but a preferential channeling of fatty acids into muscle rather than liver at high fat diets compared to wildtype. Evidence for increased lipogenesis in white fat of HSA-mUCP1 mice suggests an increased energy dissipating substrate cycling. We conclude that skeletal muscle mitochondrial uncoupling does not protect from the development of obesity in all circumstances. Rather it can lead to a "healthy" obese phenotype by preserving insulin sensitivity and a high metabolic flexibility and thus protecting from the development of obesity associated disturbances of glucose homeostasis.