Liposomes were prepared from a milk fat globule membrane (MFGM) phospholipid fraction and from soy phospholipid material using a high-pressure homogenizer (Microfluidizer). The liposomes were characterized in terms of general structure, phase transition temperature, lamellarity, bilayer thickness, and membrane permeability. The liposomes prepared from the MFGM fraction had a significantly higher phase transition temperature, thicker membrane, and lower membrane permeability. These differences were attributed to different phospholipid compositions of the MFGM and soy phospholipid fractions.