Dexamethasone is routinely used for treating those mothers at risk for preterm delivery. However, overexposure to exogenous glucocorticoids induces bone loss in offspring, and the “critical window” and safe dose of this treatment are largely unknown. In this study, we found that femoral length, and the length of the primary ossification center were significantly reduced in fetal mice after repeated prenatal dexamethasone exposure (PDE). Compared with single-course exposure on gestational day (GD)15, newborn mice with repeated PDE (3 times, from GD15 to 17) showed a significant decrease in femoral trabecular bone mass with decreased trabecular number and thickness. For those newborn mice treated after repeated PDE at different doses (0, 0.2, 0.8, and 1.2mg/kg/d), the toxic effect of dexamethasone on bone development was observed at 0.8 and 1.2mg/kg/d. More severe retardation in bone development was observed in the fetal mice after PDE at 0.8mg/kg/d during GD12–14, compared with that during GD15–17. Interestingly, stronger toxic effects were observed in male newborn mice after PDE than were observed in female newborn mice. In conclusion, PDE with multiple course, higher dose, or exposure at an early stage of pregnancy have stronger toxic effects on bone development of fetal mice.