Intrauterine malnutrition is associated with increased susceptibility to chronic diseases in adulthood. Growth-restricted infants display a less favorable lipid profile already shortly postnatal. Maternal low protein diet (LPD) during gestation is a well-defined model of fetal programming in rodents and affects lipid metabolism of the offspring. Effects of LPD throughout gestation on physiologic relevant parameters of lipid metabolism are unclear. We aimed to determine effects of LPD on maternal-fetal cholesterol fluxes and fetal lipid synthesis in mice. Pregnant mice (dams) were fed with a control (18% casein) or an LPD (9% casein) from E0.5 onward. We quantified maternal-fetal cholesterol transport and maternal cholesterol absorption at E19.5 using stable isotopes. We determined fetal lipid biosynthesis at E19.5, after administration of (1-C)-acetate from E17.5 onward. LPD did not change fetal and maternal plasma and hepatic concentrations of cholesterol and triglycerides. LPD affected neither the magnitudes of maternal-fetal cholesterol flux, maternal cholesterol absorption, nor fetal synthesis of cholesterol and palmitate (both groups, approximately 14% and approximately 13%, respectively). We conclude that LPD throughout gestation in mice does not affect maternal-fetal cholesterol transport, fetal cholesterol or fatty acid synthesis, indicating that programming effects of LPD are not mediated by short-term changes in maternal-fetal lipid metabolism.