Prolonged exposure to elevated glucocorticoid levels is known to produce insulin resistance (IR), a hallmark of diabetes mellitus. Although not fully elucidated, the underlying molecular mechanisms by which glucocorticoids induce IR may provide potential targets for pharmacological interventions. Here, we characterized muscle lipid metabolism in a dexamethasone-aggravated diet-induced obesity (DIO) murine model of IR. Male C57BL/6 mice on a high fat diet for two months when challenged with dexamethasone, showed elevated food consumption and weight gain relative to age and diet-matched animals dosed with saline only. Dexamethasone treatment impaired glucose tolerance while significantly increasing the intramyocellular lipid (IMCL) content in the Tibialis anterior muscle (TA). A good correlation (r=0.76, p<0.01) was found between IMCL_TA accumulation and visceral adiposity. The linoleic acid (18:2) to polyunsaturated acid (PUFA) ratio was increased in the dexamethasone-treated animals (+29%; p<0.01), suggesting a possible increase in Stearoyl-CoA Desaturase 2 (SCD2) activity, as reported in Sertoli cells. The treatment was also accompanied by a reduction in the % fraction of -3 and long-chain polyunsaturated fatty acids in the TA. Analysis of the low molecular weight metabolites from muscle extracts showed that there was no dysregulation of muscle amino acids, as has been associated with dexamethasone-induced muscle proteolysis. In conclusion, dexamethasone induced-insulin resistance in DIO mice is associated with a profound perturbation of lipid metabolism. This is particularly true in the muscle, where an increased uptake of circulating lipids along with a conversion into diabetogenic lipids can be observed.