Milk fat is secreted in a unique structure, termed milk fat globule (MFG) which consists of a triglyceride core covered with 3 layers of phospholipids (MFG membrane; MFGM). MFG are secreted in a wide range of sizes; from the nanometer length scale to over 15 μm, and their size is tightly associated with their lipid composition. Particularly, higher MFGM content is found in small compared with large globules. MFG size is determined by the size of its precursors — the intracellular lipid droplets (LD) which are produced and secreted by the mammary epithelial cells (MEC). Fusion is one of the suggested mechanisms controlling LD size. Nevertheless, what controls the extent of fusion and how dominant this mechanism is in controlling LD size is still illusive, especially in mammalian cells. We hypothesized that LD fusion is controlled by the stability of their membrane, which is modulated by the content and mass ratio between 2 main phospholipids - phosphatidylethanolamine (PE) and phosphatidylcholine (PC). We used primary MEC culture, treated with oleic or palmitic acid, to study the role of membrane stability in determining LD size. Results show that 22% of MEC treated with oleic acid had large LD (>2.5 µm) compared with only 4% of the cells treated with palmitic acid. The increased LD size in the oleic acid treatment was associated with 63% increase in PE, and 7 fold increase in LD fusion. Adding NaN₃+NaF to the oleic acid treatment decreased PE content by 19%, concomitantly with 8 fold decrease in the number of large LD. Interestingly, the addition of NaN₃+NaF to oleic acid treatment did not change the cellular triglyceride content. In contrast, adding 3-deazaadenosine to palmitic acid treatment tended to increase PE content by 29%, and consequently increased the number of large LD by 3 fold, relative to cells treated with palmitic acid alone. Our findings have uncovered a defining role for LD fusion in determining their size in MEC, which is independent of triglycerides content of the cells. Understanding the mechanisms controlling LD size in mammalian cells is of great importance, especially in MEC due to the effect of LD size on milk composition.