We recently reported insulin resistance in adult offspring of obese C57Bl/6J mice. We have now evaluated whether parameters of skeletal muscle structure and function may play a role in insulin resistance in this model of developmental programming. Obesity was induced in female mice by feeding a highly palatable sugar and fat-rich diet for 6 weeks prior to pregnancy, and during pregnancy and lactation. Offspring of obese dams were weaned onto standard laboratory chow. At 3 months of age, skeletal muscle insulin signaling protein expression, mitochondrial electron transport chain activity (ETC), muscle fiber type, fiber density and fiber cross sectional area were compared with that of offspring of control dams weaned onto the chow diet. Female offspring of obese dams demonstrated decreased skeletal muscle expression of p110, the catalytic subunit of PI3K (p<0.01) as well as reduced Akt phosphorylation at Serine residue 473 when compared to control offspring. Male offspring of obese dams demonstrated increased skeletal muscle Akt2 and PKC expression (p<0.01; p<0.001 respectively). A decrease in mitochondrial-linked complex II-III was observed in male offspring of obese dams (p<0.01), which was unrelated to CoQ deficiency. This was not observed in females. There were no differences in muscle fiber density between offspring of obese dams and control offspring in either gender. Gender related alterations in key insulin signaling proteins and in mitochondrial ETC may contribute to a state of insulin resistance in offspring of obese mice.