Background: Mammary gland(MG) de-novo lipogenesis contributes significantly to milk fat in animals but little is known in humans. Objective: To test the hypothesis that the incorporation of ¹³C carbons from [U-¹³C]glucose into fatty acids (FA) and glycerol in triglycerides (TG)will be greater: a)in milk than plasma TG, b)during a high carbohydrate (H-CHO) diet than high fat (H-FAT) diet and c)during feeding than fasting. Materials/Methods: Seven healthy lactating women were studied on two isocaloric, isonitrogenous diets. On one occasion subjects received diets containing H-FAT or H-CHO diet for 1 week. Incorporation of ¹³C from infused [U-¹³C]glucose into FA and glycerol was measured using GC/MS and gene expression in RNA isolated from milk fat globule using microarrays. Results: Incorporation of ¹³C₂ into milk FA, increased with increased FA chain length from C2:0 to C12:0 but progressively declined in C14:0 and C16:0 and was not detected in FA>C16. During feeding, regardless of diets, enrichment of ¹³C₂ in milk FA and ¹³C₃ in milk glycerol were ~3 and ~7 fold higher compared to plasma FA and glycerol, respectively. Following an overnight fast during H-CHO and H-FAT diets, 25% and 6%, respectively, of medium chain FA (MCFA, C6-C12) in milk were derived from glucose but increased to 75% and 25% with feeding. Expression of genes involved in FA or glycerol synthesis was unchanged regardless of diet or fast/fed conditions. Conclusions: The human MG is capable of de novo lipogenesis, of primarily MCFA and glycerol, which is influenced by the macronutrient composition of the maternal diet.