Milk fat is secreted mainly in the form of fat globules (MFG) from the mammary glands of female mammals into the milk. The MFG particle size ranges from 0.2 to 15.0 μm. MFG sizes can affect the health of newborns and the physical and chemical properties of dairy products. As the size of MFG is determined by intracellular lipid droplets during the secretion of mammary epithelial cells, the regulation factors of lipid droplet size include intracellular triglyceride (TG) level and enzymes involved in the synthesis and hydrolysis of TG, enzyme, membrane phospholipid, and lipid droplet fusion, etc. This review mainly describes the synthesis and structure of MFG, the regulation of MFG particle size and its impact on human nutrition, dairy processing and lipid droplet size fusion, which will provide reference for the application of milk fat globule size.