The World Health Organization recommends exclusive breastfeeding for the first 6 months of life. Breastfeeding is known to have beneficial effects on numerous physiological functions, such as the maturation of the newborn's intestinal barrier. Epidemiological studies also show the benefits of breastfeeding duration in preventing short- and medium-term cardio-metabolic pathologies in offspring. In parallel, several studies suggest an adaptation of milk composition (colostrum or mature milk) associated with a context of maternal gestational diabetes (GDM). Taken together, these data lead us to hypothesize that, in a context of GDM, the composition of milk, and in particular its content of bioactive compounds regulating energy homeostasis, would adapt to this maternal hyperglycemic environment and could have a potentially beneficial effect on the prevention of cardio-metabolic diseases in the offspring. To test this hypothesis, we developed a nutritional model of DG rats. Female rats were fed a high-fat, high-sucrose diet (HFHS) or a standard diet (CTL) one week before and during gestation. During lactation, the HFHS group was fed a standard diet (4HFHS) or maintained on HFHS (7HFHS). We calculated the area under the curve (AUC) and an insulin sensitivity index (ISIGutt) following oral glucose tolerance tests (OGTT) performed during gestation (G12) and lactation (L12). Milk composition was characterized on day 1 of lactation (L1), then at L8, L14 and L19 by targeted analysis of total fatty acids (gas chromatography), amino acids and sphingolipids, as well as by high-throughput analysis of lipidomic profiles (liquid chromatography/tandem mass spectrometry). At G12, rats in the HFHS group showed significantly increased insulin and blood glucose levels (AUC) and a reduced ISIGutt index vs. the CTL group. At L12, AUC blood glucose was increased and ISIGutt reduced in 7HFHS vs. 4HFHF and CTL rats. In this context of maternal hyperglycemia, 7HFHS milk showed throughout lactation, vs. CTL milk, a significant increase in the concentration of insulin-trophic amino acids, an increase in the ratio of ω6/ω3 polyunsaturated fatty acids, as well as altered profiles in certain lipid species known to interfere with insulin sensitivity (2-factor ANOVA). hese differences in breast milk composition were also significant at L1 in 4HFHS vs CTL milk. Finally, at L1, milk from 4HFHS and 7HFHS rats showed a different lipidomic signature from milk from CTL mothers (Partial Least Squares Discriminant Analysis - PLS-DA). These results suggest an adaptation of milk composition during lactation associated with maternal hyperglycemia during gestation and/or lactation. Longitudinal follow-up of offspring born to CTL or DG mothers and adopted by euglycaemic and/or hyperglycaemic mothers is currently being analysed to determine whether this adaptation of milk composition to DG is associated with reduced cardiometabolic risks for offspring in the long term.