Antenatal glucocorticoids are administered to women at risk of preterm delivery to prevent neonatal respiratory morbidity. The effects of exogenous glucocorticoids on the development of lactation are unknown. This study investigated the effects of a single dose of antenatal glucocorticoids on secretory activation in sheep before and after parturition. Pregnant ewes (N=36) were randomised to receive either medroxyprogesterone acetate (MPA) at 118 days of pregnancy and betamethasone at 125 days (BETA group), MPA at 118 days and saline at 125 days (MPA group) or saline at 118 and 125 days (SALINE group). The concentration of lactose, progesterone, cortisol and prolactin in maternal plasma was measured during pregnancy. After term parturition, the concentration of lactose in milk and maternal plasma was measured daily for 5 days. Lambs were weighed at birth and at 5 days of age; milk volume was measured on day 5. The concentration of lactose in maternal plasma increased significantly after betamethasone administration, corresponding to a fall in plasma progesterone. No changes in lactose were observed in MPA or SALINE ewes. Transient decreases in cortisol and increases in prolactin were observed in the BETA group, but not in either the MPA or SALINE group. After parturition, BETA ewes experienced reduced milk yield and lamb weight gain, and delayed increases in milk lactose levels compared with MPA and saline controls. This study demonstrated that, in sheep, antenatal glucocorticoid administration disrupted secretory activation, causing precocious mammary secretion before parturition and compromising postpartum milk production and lamb growth.