Dexamethasone is used as treatment for a variety of neonatal syndromes, including respiratory distress. The present study utilized the power of comprehensive lipid profiling to characterize changes in lipid metabolism in the neonatal lung and brain associated with dexamethasone treatment, and also determined the interaction of dexamethasone with hypoxia. A four-day tapering dose regimen of dexamethasone was administered on post-natal day 3 (PD3; 0.5 mg/kg), PD4 (0.25 mg/kg), PD5 (0.125 mg/kg), and PD6 (0.05 mg/kg). A subgroup of rats was exposed to hypoxia from birth to seven days of age. Dexamethasone treatment elicited numerous specific changes in the lipid profile of the normoxic lung, such as increased concentrations of saturated fatty acids in the phosphatidylcholine and cholesterol ester classes. These increases were more profound in the lungs of hypoxic pups. Additional increases in cardiolipin concentrations were also measured in lungs of hypoxic pups treated with dexamethasone. We measured widespread increases in serum lipids following dexamethasone treatment, but the effects were not equivalent between normoxic and hypoxic pups. Dexamethasone treatment in hypoxic pups increased 20:4n6 and 22:6n3 concentrations in the free fatty acid class of the brain. Our results suggest that dexamethasone treatment in neonates elicits specific changes in lung lipid metabolism associated with surfactant production, independent of changes in serum lipids. These findings illustrate the benefits of dexamethasone on lung function, but also raise the potential for negative effects due to hyperlipidemia and subtle changes in brain lipid metabolism.