Breast milk contains macromolecular components with distinctive functions, whereby milk fat globules and casein micelles provide nutrition, while whey contains molecules that stimulate the infant’s immunity. Interestingly, the importance and function of extracellular vesicles (EV) in milk has not been addressed in detail. This in part is due to the difficulty to purify EV from other milk components and consequently the molecular composition of human milk-derived EV has not been fully elucidated. Methods: In this study, an extensive LC-MS/MS proteomic analysis was performed on EV isolated via density-based separation from human breast milk of 7 individuals. The proteome of milk-derived EV was analyzed using FunRich and compared to the Vesiclepedia database, as well as to the whole milk proteome, which was constructed from 38 previously published milk proteomics studies.