Technical Abstract: Exosomes are 40-100 nm membrane vesicles of endocytic origin and are found in blood, urine, amniotic fluid, bronchoalveolar lavage (BAL) fluid, as well as human and bovine milk. Exosomes are extracellular organelles important in intracellular communication/signaling, immune function, and biomarkers for disease. Therefore, the proteome of bovine mammary-derived exosomes in milk is of considerable interest as milk exosomes have potential immune functions important in intramammary infections. Furthermore, these milk exosomes may provide insight into the complex processes of milk production. Triplicate exosome isolations from the milk of 2 mid-lactation cows were trypsin digested, subjected offline high pH reverse phase chromatography further fractionated on a nanoLC connected to tandem mass spectrometer. Analysis of the data resulted in identification of 2107 proteins in milk exosomes. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactaherin) were among the most abundant proteins found in milk exosomes. They represented only 0.4-1.2% of the total spectra collected compared to 15-28% of the total spectra collected in the MFGM proteome. This indicates that the milk exosome pathway through the secretory cell apical plasma membrane into milk differs significantly from that of the MFGM. The protein composition of milk exosomes provides new information as to the potential physiological significance of exosomes to mammary physiology, health, and calf nutrition.