Background: In pregnant women with gestational diabetes, glyburide can be an alternative to insulin despite concerns about its transplacental transfer. However, transplacental transfer of glyburide is poorly quantified and the relationship between cord blood glyburide concentration and hypoglycemia has not been studied. Our objective was to quantify the transplacental transfer of glyburide at delivery and to study the association between the cord blood glyburide concentration and the risk of neonatal hypoglycemia in patients with gestational diabetes treated with glyburide. Methods and findings: INDAO was a multicenter, noninferiority, randomized trial conducted between May 2012 and November 2016 in 914 women with singleton pregnancies and gestational diabetes. An ancillary study was conducted in the 87 patients of the Bicêtre University Hospital Center. The sample consisted of 46 patients with utilizable assays at delivery. The relationships between glyburide concentration and the time since the last intake of glyburide and between fetal glyburide concentration and neonatal hypoglycemia were modeled with linear or logistic regressions using fractional polynomials. There was placental transfer of glyburide at a fetal to maternal ratio of 62% (95% CI [50; 74]). Umbilical cord blood glyburide concentration decreased steeply after the last maternal glyburide intake. After 24 hours, the mean umbilical cord blood concentration was less than 5 ng/mL. Neonatal hypoglycemia risk was increased with an odds ratio of hypoglycemia equal to 3.70 [1.40-9.77] for each 10 ng/mL increase in the cord blood glyburide concentration. However, no newborns were admitted to the NICU because of clinical signs of hypoglycemia or for treatment of hypoglycemia. Conclusion: Considering that neonatal glyburide exposure may be limited by stopping treatment a sufficient time before labor, there may still be a place for glyburide in the management of gestational diabetes.