Milk fat globules (MFG) are secreted by mammary epithelial cells (MEC) into mammary gland alveolar lumen in a wide range of sizes, from 200 nm to over 15 µm. The significance of size derives from its tight association with MFG lipidome and proteome. Moreover, small MFG have relatively higher content of membrane compared with large globules, and this membrane exerts diverse positive health effects, as reported in human and animal studies. In addition, MFG size has industrial relevance since it affects the physicochemical and sensory characteristics of dairy products. MFG size is determined before and during its secretion from MEC, and therefore the size of their precursors, the intracellular lipid droplets, will determine the MFG diameter. Two main mechanisms regulate the size of lipid droplets; the first is based on intracellular triglyceride content, regulated by metabolic signals. In various lipogenic tissues, like adipose and liver, anabolic signals induce triglyceride storage and hence increase lipid droplet size. However, the positive association between triglyceride level and lipid droplet size in MEC and milk does not always hold. Interestingly, MFG size is usually elevated under conditions of negative energy balance, when catabolic signals reduce insulin and elevate NEFA plasma concentrations. Therefore, triglyceride synthesis and secretion capacity cannot in itself explain the differences in MFG size and a second mechanism, fusion, was proposed to contribute to MFG size regulation. Fusion between lipid droplets occur in MEC cytoplasm, as was demonstrated in murine MEC in vivo. However, the regulatory mechanisms of fusion are still illusive. We showed that membrane phospholipid composition, partially determined by mitochondria enzymes, can change membrane stability and hence control the extent of lipid droplet fusion and consequently, MFG size. This regulation is independent of the triglyceride content in the cells. The fact that mitochondria play a role in size regulation, independently of fat production, may explain the size variations occur during lactation, concomitantly with changes in metabolic status of the animal, as often demonstrated in dairy cows.