Background: Expression of genes for lipid biosynthetic enzymes during initiation of lactation in humans is unknown. Objective: To study mRNA expression of lipid metabolic enzymes in human mammary epithelial cell (MEC) in conjunction with the measurement of milk fatty acid (FA) composition during secretory activation. Subjects: Gene expression from mRNA isolated from milk fat globule (MFG) and milk FA composition were measured from 6h to 42days postpartum in 7 normal women. Results: Over the first 96h postpartum, daily milk fat output increased several fold and mirrored expression of genes for all aspects of lipid metabolism and milk FA production including: lipolysis at the MEC membrane, FA uptake from blood, intracellular FA transport, de novo FA synthesis, FA and glycerol activation, FA elongation, FA desaturation, triglyceride synthesis, cholesterol synthesis, and lipid droplet formation. Expression of the gene for a key lipid synthesis regulator, sterol regulatory element-binding transcription factor1 (SREBF1), increased 2.0 fold by 36h and remained elevated over the study duration. Expression of genes for estrogen receptor1 (ESR1), thyroid hormone responsive protein (THRSP) and insulin-induced2 (INSIG2) increased progressively to plateau by 96h. In contrast, mRNA of peroxisome proliferator-activated receptor gamma (PPARG) decreased several fold. With the onset of lactation, increased de novo synthesis of FAs was the most prominent change in milk FA composition and mirrored the expression of FA synthesis genes. Conclusion: Milk lipid synthesis and secretion in humans is a complex process requiring the orchestration of a wide variety of pathways of which SREBF1 may play a primary role.