Exosomes and ectosomes in general were considered fragments derived from death cells; however, it is now known their participation in short and long communication between cells. Even though they have different origin since exosomes have intracellular biogenesis and ectosomes are assembled from the cell plasma membrane, these milk micro-vesicles share molecular features between each other and with the MFGM such as protein composition; however, not much has been addressed regarding lipid composition, vesicle size and distribution, or surface characteristics (Z-potential). The aim of this study was to isolate the micro-vesicles from raw milk fractions by ultracentrifugation (110,000 xg) on a sucrose cushion (30%) and perform the characterization of the different vesicles based on their colloidal behavior in solution (reconstituted in PBS buffer). The components in each fraction were isolated by SEC and subjected to dynamic light scattering (NanoBrook 90 PlusPALS, Brookheaven Instruments) collecting the dispersed light at angles of 90° and 15° to resolve isolated particles from large aggregates. We found significant differences between the size of MFGM particles, exosomes (ranging from 50 to 100 nm) and ectosomes (ranging from 100 to 350 nm). We also determined lipid composition of each fraction by extracting lipids (Bligh and Dyer methodology) and analyzing them by liquid chromatography on an HPLC system coupled to a charged aerosol detector (Corona Veo RS, Thermo Scientific). Even though every fraction is composed of typical phospholipids (phosphatidylcholine, phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine), cholesterol and sphingomyelin found in milk, their distribution in each group of vesicles is different, thus, correlating with the different values of surface Z-potential determined in solution. The characterization and differentiation of milk extracellular micro-vesicles derived from this work, along with the protein and nucleotide composition already reported elsewhere, will help understand the function of these structures during digestion, and as transporters of biological active agents with promising application in human therapy diseases