Antenatal administration of betamethasone (BM) is a common antecedent of preterm birth, but there is limited information about its impact on the acute evolution of preterm neonatal brain injury. We aimed to compare the effects of maternal BM in combination with mechanical ventilation on the white matter (WM) of late preterm sheep. At 0.85 of gestation, pregnant ewes were randomly assigned to receive intra-muscular (i.m.) saline (n = 9) or i.m. BM (n = 13). Lambs were delivered and unventilated controls (UVC Sal , n = 4; UVC BM, n = 6) were humanely killed without intervention; ventilated lambs (Vent Sal , n = 5; Vent BM , n = 7) were injuriously ventilated for 15 min, followed by conventional ventilation for 75 min. Cardiovascular and cerebral haemodynamics and oxygenation were measured continuously. The cerebral WM underwent assessment of inflammation and injury, and oxidative stress was measured in the cerebrospinal fluid (CSF). In the periventricular and subcortical WM tracts, the proportion of amoeboid (activated) microglia, the density of astrocytes, and the number of blood vessels with protein extravasation were higher in UVC BM than in UVC Sal (p < 0.05 for all). During ventilation, tidal volume, mean arterial pressure, carotid blood flow, and oxygen delivery were higher in-Vent BM lambs (p < 0.05 vs. Vent Sal ). In the subcortical WM, microglial infiltration was increased in the Vent Sal group compared to UVC Sal . The proportion of activated microglia and protein extravasation was higher in the Vent BM group compared to Vent Sal within the periventricular and subcortical WM tracts (p < 0.05). CSF oxidative stress was increased in the Vent BM group compared to UVC Sal, UVC BM , and Vent Sal groups (p < 0.05). Antenatal BM was associated with inflammation and vascular permeability in the WM of late preterm fetal sheep. During the immediate neonatal period, the increased carotid perfusion and oxygen delivery in BM-treated lambs was associated with increased oxidative stress, microglial activation and microvascular injury.