The molecular physiology underlying human milk production is largely unknown because of limitations in obtaining tissue samples. Determining gene expression in normal lactating women would be a potential step toward understanding why some women struggle with or fail at breastfeeding their infant. Recently, we demonstrated the utility of RNA obtained from breast milk fat globule (MFG) to detect mammary epithelial cell (MEC)-specific gene expression. We used MFG RNA to determine the gene expression profile of human MEC during lactation. Microarray studies were performed using Human Ref-8 BeadChip arrays (Illumina, Inc). MFG RNA was collected every 3h for 24h from 5 healthy, exclusively breastfeeding women. 14,070 transcripts were expressed and represented the MFG transcriptome. Using GeneSpring GX 9, 156 ontology terms (corrected P < 0.05), which include cellular (n = 3,379 genes) and metabolic (n = 2,656) processes as the most significantly enriched biologic process terms. The top networks and pathways were associated primarily with cellular activities most likely involved with milk synthesis. Multiple sampling over 24h enabled us to demonstrate core circadian clock gene expression and the periodicity of 1029 genes (7%) enriched for molecular functions involved in cell development, growth, proliferation, and cell morphology. In addition, we found that the MFG transcriptome was comparable to the metabolic gene expression profile described for the lactating mouse mammary gland. This paper is the first to describe the MFG transcriptome in sequential human samples over a 24h period, providing valuable insights into gene expression in the human mammary epithelial cell.