Clinical safety of consuming plant stanol ester spreads during pregnancy and lactation, the impact on maternal and infant serum and breast-milk cholesterol and the ratios (μmol/mmol of cholesterol) of synthesis and absorption markers were evaluated. Pregnant women (n 21) were randomised to control and dietary intervention groups, the intervention including advice to follow a balanced diet and to consume spreads enriched with plant stanol esters. Participants were followed during and after pregnancy and their infants up to 1 year of age. A mean 1·1 (sd 0·4) g consumption of plant stanols during pregnancy and 1·4 (sd 0·9) g 1 month post-partum increased sitostanol and the markers for cholesterol synthesis, lathosterol, lathosterol/campesterol and lathosterol/sitosterol, and reduced a marker for cholesterol absorption, campesterol, in maternal serum. In breast milk, desmosterol was lower in the intervention group, while no differences were detected between the groups in infants' serum. Plant stanol ester spread consumption had no impact on the length of gestation, infants' growth or serum β-carotene concentration at 1 and 6 months of age, but the cholesterol-adjusted serum β-carotene concentration was lowered at 1 month in the intervention group. Plant stanol ester spread consumption appeared safe in the clinical setting, except for potential lowering of infants' serum β-carotene concentration, and was reflected in the markers of cholesterol synthesis and absorption in mothers' serum, encouraging further studies in larger settings.