Gestational diabetes mellitus is a major complication of human pregnancy. The oral clearance (CL) of glyburide, an oral antidiabetic drug, increases 2-fold in pregnant women during late gestation versus nonpregnant controls. In this study, we examined gestational age-dependent changes in maternal-fetal pharmacokinetics (PK) of glyburide and metabolites in a pregnant mouse model. Nonpregnant and pregnant FVB mice were given glyburide by retro-orbital injection. Maternal plasma was collected over 240 minutes on gestation days (gd) 0, 7.5, 10, 15, and 19; fetuses were collected on gd 15 and 19. Glyburide and metabolites were quantified using high-performance liquid chromatography-mass spectrometry, and PK analyses were performed using a pooled data bootstrap approach. Maternal CL of glyburide increased approximately 2-fold on gd 10, 15, and 19 compared with nonpregnant controls. Intrinsic CL of glyburide in maternal liver microsomes also increased as gestation progressed. Maternal metabolite/glyburide area under the curve ratios were generally unchanged or slightly decreased throughout gestation. Total fetal exposure to glyburide was <5% of maternal plasma exposure, and was doubled on gd 19 versus gd 15. Fetal metabolite concentrations were below the limit of assay detection. This is the first evidence of gestational age-dependent changes in glyburide PK. Increased maternal glyburide clearance during gestation is attributable to increased hepatic metabolism. Metabolite elimination may also increase during pregnancy. In the mouse model, fetal exposure to glyburide is gestational age-dependent and low compared with maternal plasma exposure. These results indicate that maternal glyburide therapeutic strategies may require adjustments in a gestational age-dependent manner if these same changes occur in humans.