Maternal obesity and gestational diabetes (GDM) are associated with adverse outcomes particularly with a male fetus. The composition and amount of substrate supplied to the placenta is altered in these conditions. We hypothesized that there are sexually dimorphic differences in utilization of glucose, fatty acids and glutamine between trophoblast of lean, obese and GDM women.Trophoblast were isolated from term male or female placentas from lean, obese or GDM women (n = 4-6/group) and syncytiotrophoblast formed over 72 hr before measuring mitochondrial respiration by fuel flex assay (Seahorse XF96 analyzer). Dependency, capacity and flexibility for use of glucose, glutamine and fatty acids was measured with western blot of glucose transporter GLUT1, glutaminase and carnitine palmitoyl-transferase 1A, (CPT1A).Sexual dimorphism in syncytiotrophoblast fuel utilization was seen in GDM vs lean with a significant increase in glucose dependency in male and glucose capacity in female, whereas for glutamine capacity significantly decreased in male and female but dependency only in female. Fatty acid dependency and capacity significantly increased in male and capacity in female trophoblast of GDM vs either lean or obese. In male but not female trophoblast flexibility to use all three fuels significantly decreased from lean to obese and GDM. In male trophoblast there were significant associations between GLUT1 and glucose dependency (positive) and flexibility (negative).Human syncytiotrophoblast utilizes glutamine for mitochondrial respiration. Utilization of glucose, fatty acids and glutamine changes in a sexually dimorphic manner with obesity and GDM predominantly with a male placenta.