Milk fat globules (MFGs) are accepted primarily as triacylglycerol delivery systems. The identification of nanometer-sized lipid-protein particles termed "lactosomes" that do not contain triacylglycerol raises the question of their possible functions. MFGs were isolated by slow centrifugation, and lactosomes were isolated by ultracentrifugation at a density equivalent to plasma high-density lipoproteins (HDL) (d > 1.063 g/mL) from human milk obtained from six volunteers at different lactation stages. Isolated lactosomes were analyzed and compared with MFGs for their size distribution, lipidome, proteome, and functional activity. Lactosomes from early milk, day 8, were found to be similar in size as those from mature milk >28 days, averaging ∼25 nm in diameter. In total, 97 nonredundant proteins were identified in the MFG and lactosome fractions, 46 of which were unique to the MFG fraction and 29 of which were unique to the lactosome fraction. The proteins identified in the lactosome and MFG fractions were enriched with proteins identified with immunomodulatory pathways. Unlike MFGs and GM1-laden reconstituted HDL that served as a positive control, lactosomal binding capacity to cholera toxin was weak. Lipidomic analyses found that lactosomes were devoid of triacylglycerol and gangliosides, unlike MFGs, but rich in a variety of phospholipid species. The data found differences in structure, composition, and function between lactosomes and MFG, suggesting that these two particles are derived from different biosynthetic and/or secretory pathways. The results reveal a bioactive lipid-protein, nanometer-length scale particle that is secreted into milk not to supply energy to the infant but to play unique, protective, and regulatory roles.