The synthesis of milk constituents in the mammary epithelial cells involves several complex and coordinated biochemical and metabolic pathways. The precursors of milk constituents are transported from blood to milk via transcellular and paracellular routes with the help of transport proteins at the apical and basolateral membranes. Glucose is the main energy source in the mammary gland. The mammary gland utilizes glucose in three ways: synthesis of lactose, synthesis of NADPH and milk fat, and nucleic acid synthesis and energy production. The mammary gland is considered the most efficient tissue to produce lipids. Dietary fatty acids are transported from the intestine to the mammary gland in the form of chylomicron, but mammary gland can also synthesize fatty acids either from acetate (ruminants) or glucose (nonruminants). These fatty acids are esterified with glycerol to form milk triglycerides. Milk proteins are synthesized from amino acids in the ribosome followed by post-translational modification in the Golgi complex. Lactose and milk proteins are packaged into secretory vesicles and released in milk by exocytosis. Milk fat globules are extruded from the apex of the secretory cell surrounded by milk fat globule membrane. RNA and pyridine nucleotides are synthesized from ribose. Immunoglobulins are transported directly into the milk through the process of transcytosis. There is notable diversity in the metabolism of the mammary gland between species that leads to species-specific milk composition.