The effect of an exogenous glucocorticoid on the lipid metabolism and fatty acid pattern of skeletal muscle in broiler chickens (Gallus gallus domesticus) was investigated in vivo and in vitro. Male Arbor Acres chickens were subjected to dexamethasone (DEX) treatment for 3days. We found that DEX retarded body growth, facilitated lipid accumulation in adipose and skeletal muscle tissues, and elevated the thigh monounsaturated fatty acids (MUFA) to saturated fatty acids (SFA) ratio at fasted state. DEX-treated chickens exhibited increased stearoyl-CoA desaturase-1 (SCD1) activity and decreased carnitine palmitoyltransferase-1 (CPT1) activity in the thigh muscle under fasting conditions and in primary cultured myoblasts. Phosphorylation of AMP-activated protein kinase alpha at Thr172 did not occur in vivo but was increased in vitro by DEX. In cells exposed to DEX, fatty acid transport protein-1 mRNA expression and fatty acid storage were enhanced while fatty acid oxidation was repressed. In conclusion, in oxidative muscle of fasted chickens, DEX stimulated uptake of myocellular fatty acids which was stored with the modified MUFA to SFA ratio in a process that maybe involved SCD1 activation. The altered fatty acid composition together with the inactivation of CPT1 showed an increased tendency towards fatty acid accumulation as opposed to oxidation. These findings provide important insight concerning the influence of glucocorticoids on lipid metabolism.