Abstract: In order to compare the differences in typical characterization of bovine milk exosomes between raw milk and liquid dairy products processed by different processing technologies, and to investigate the effects of production process and gastrointestinal digestion on the characterization and transport capacity of bovine milk exosomes, this study selected raw milk, commercial pasteurized milk and ultra-high temperature instantaneous sterilized (UHT) milk as research subjects. The gastrointestinal digestion process was simulated using the international INFOGEST in vitro digestion model. Bovine milk exosomes were isolated using isoelectric precipitation combined with ultracentrifugation. Comprehensive characterization of exosomes was performed by measuring total protein concentration with a BCA protein assay kit, observing morphology by transmission electron microscopy, analyzing particle size distribution by nanoparticle tracking analysis, and detecting specific protein markers (CD9, CD63, and TSG101) by western blotting. Additionally, the uptake of exosomes by bovine mammary epithelial cells (MAC-T) and rat intestinal epithelial cells (IEC-6) was visualized by confocal laser scanning microscopy. The results showed that the total protein concentration of exosomes isolated from equal volumes of raw, pasteurized and UHT milk increased sequentially with significant differences (P<0.05). Exosomes derived from raw milk exhibited significantly larger particle sizes than those derived from pasteurized and UHT milk (P<0.05), whereas no regular pattern was observed in particle size changes induced by simulated gastrointestinal digestion. Both production process and gastrointestinal digestion reduced the integrity of exosomal surface proteins to varying degrees, with UHT milk exosomes showing only weak expression of TSG101 and CD63. All three specific protein markers became undetectable after digestion. Fluorescent tracer experiments revealed no significant differences in the uptake efficiency of bovine milk exosomes by two target cell types (IEC-6 and MAC-T), and exosomes subjected to processing or digestion were still non-specifically internalized by these cells. This study demonstrated that production process and gastrointestinal digestion significantly compromised the characteristic properties of bovine milk exosomes but did not entirely abolish their transport capacity, providing a theoretical foundation for the development of functional dairy products.