The MFGM is a very complex component in milk that provides phospholipids, including sphingomyelin, which are of great interest in human nutrition and have demonstrated biological activity in intestinal cells and more recently in early brain development. It is structured in a specific highly organized architecture in raw milk. After processing, we assume that this architecture/structure in the MFGM is a good model for developing functional dairy foods. MFGM and its components possess an interesting technological function as an ingredient. However, there is little information regarding the microstructure, physical and chemical features of highly pure MFGM phospholipid powders after hydration. In this work, we aimed to reconstitute and characterize MFGM phospholipids into liposomes (800 nm diameter) to investigate structural features upon hydration by confocal fluorescence microscopy. We also determined their physicochemical parameters such as z-potential by dynamic light scattering. Additionally, we conducted stability studies calculating the changes in the Tm value by fluorescence-based thermal shifts using SYPRO Orange dye as the fluorescent probe. A specific mixture of milk phospholipids kindly donated by Fonterra Co-Op, NZ was used to produce our vesicles by extrusion of a thin film. We have found that MFGM reconstituted liposomes (1.5 mg/mL in PBS buffer pH 7.4) associate into unique vesicles generating a binary mixture of liposomes (250 and 800 nm diameter) with a charge in colloidal solution of −21.5 mV indicating that negatively charged phospholipids are assembled predominantly in the surface of the liposomes.