Better knowledge on the disturbed mechanisms implicated in materno-fetal long-chain polyunsaturated fatty acid (LC-PUFA) transfer in pregnancies with gestational diabetes mellitus (GDM) may have potential high implications for later on in effective LC-PUFA supplementation. We studied in vivo placental transfer of fatty acids (FA) using stable isotopes tracers, administrated to 11 control and 9 GDM pregnant women (6 treated with insulin). Subjects received orally ¹³C-palmitic, ¹³C-oleic and 13C-linoleic acids and 13C-docosahexaenoic acid (¹³C-DHA) 12h before elective caesarean section. Maternal blood samples were collected at -12,-3,-2,-1, delivery and +1h. Placental tissue and venous cord blood were also collected. FA were quantified by gas chromatography (GC) and ¹³C-enrichments by GC-isotope ratio mass spectrometry. ¹³C-FA concentration was higher in total lipids of maternal plasma in GDM vs. controls, except for ¹³C-DHA. Moreover, 13C-DHA showed lower placenta/maternal plasma ratio in GDM vs. controls and significantly lower cord/maternal plasma ratio. For the other studied FA ratios were not different between GDM and controls. Disturbed ¹³C-DHA placental uptake occurs in both GDM treated with diet or insulin, while the last ones also have lower ¹³C-DHA in venous cord. The tracer study pointed towards impaired placental DHA uptake as critical step, while the transfer of the rest of ¹³C-FA was less affected. GDM under insulin treatment could have also higher fetal fat storage contributing to reduce ¹³C-DHA in venous cord. DHA transfer to the fetus was reduced in GDM pregnancies compared to controls which might affect the programming of neurodevelopment in their neonates.