Midazolam is used in pregnancy post the second trimester for the treatment of convulsive seizures or as an anesthetic during cesarean section. However, its safety has not been validated. If the migration and accumulation of midazolam into the fetal brain can be clarified, the extent of damage to the fetal brain can be predicted. Therefore, we investigated the migration of midazolam and its active metabolite 1′-hydroxymidazolam into the fetal brain. Midazolam was administered intravenously to pregnant mice in the second trimester (E14.5), and the migration of midazolam and 1′-hydroxymidazolam to the maternal brain and plasma as well as the fetal brain were analyzed over time. Fetal brain levels of midazolam and 1′-hydroxymidazolam were high at 44.7% and 44.5% of that in the maternal brain. Furthermore, both mothers and fetuses expressed 1:1 brain ratios of midazolam and 1′-hydroxymidazolam. In both mothers and fetuses, migration of midazolam into the brain was higher than the migration of 1′-hydroxymidazolam. In this study, approximately half of midazolam and 1′-hydroxymidazolam in the maternal brain was transferred to the fetal brain. During the second trimester, neural stem cells in the fetal brain differentiate into neurons and glial cells and higher brain functions are developed. In the present study, we observed increased migration of midazolam and 1′-hydroxymidazolam into the fetal brain during this period. Therefore, damage to neuronal development may still be observed in the fetus even with post-second trimester midazolam use.