Lipid droplets synthesized in mammary epithelial cells are secreted into breast milk by the budding-off mechanism. The milk lipids, termed mik fat globules (MFGs), are surrounded with the cell plasma membrane and contain various membrane proteins, including milk fat globule epidermal growth factor (EGF)-factor VIII (MFG-E8), on their surface. We report here that the MFGs in the milk of MFG-E8-deficient mice fused each other and turned into abnormally large size of lipid droplets within ∼48 h after being secreted into mammary alveolar lumen in situ or being incubated at 37°C in vitro. This biophysical degeneration of MFGs in the MFG-E8-deficient milk was efficiently rescued in vitro by adding the milk serum of wild-type mice, isolated MFG-E8 or annexin V. Moreover, addition of ethylenediaminetetraacetic acid (30 mM) also protected the MFG fusion remarkably in vitro. In addition, bovine MFGs also fused each other when isolated from milk serum, and the fusion was inhibited by adding isolated MFG-E8 or mouse milk serum, but not the milk serum of MFG-E8-deficient mice. MFG-E8 in breast milk may mask the phosphatidylserine exposed on the surface of MFGs with time after secretion and thereby suppress the membrane fusion among MFGs resulting in the enlargement of MFGs in the breast milk.