Glucose is an essential nutrient for the conceptus. The objective was to determine if lactation affected the amount of glucose crossing the placenta by measuring glucose and fructose in placental fluids in lactating and nonlactating cows. Holstein cows were assigned to one of 2 treatments immediately after parturition [lactating (n=23) or nonlactating (dried off immediately after calving; n=20)]. Pregnant cows were slaughtered at one of 3 d of pregnancy (d 28, 35, or 42) and tissues were collected. Plasma glucose and insulin were less in lactating cows. Pregnancies collected from lactating cows had less glucose and fructose in placental fluids compared with those from nonlactating cows. Relative to endometrium, the placenta expressed greater amounts of the glucose transporters SLC2A1 (Glut1), SLC2A3 (Glut3) and SLC2A4 (Glut4) mRNA. The mRNA for SLC2A1 decreased whereas the mRNA for SLC2A4 increased from d 28 to d 42 of pregnancy. Stepwise regression analyses for fetal and placental weight (dependent variable) retained day of pregnancy and maternal plasma insulin concentrations in the final model. The conclusion is that lower blood glucose and insulin in lactating cows may lead to less glucose crossing the placenta and slower fetal development during lactation. The slower fetal development may predispose lactating cows to fetal loss if developmental milestones are not reached.