Updated project metadata. Exosomes are nanosized membranous vesicles secreted by variety types of cells and regulate intercellular communication through deliver bioactive substances. They are found in abundance in biological fluids including semen. Although protein composition and potential fertility biomarkers of seminal plasma exosomes (SPE) have been identified in human, but they remains unclear in buffalo. Here, we aim to discover the protein correlation between spermatozoa, seminal plasma (SP) and SPE, then further compare and analyze the protein differences between high and low motility SPEs in buffalo. SPEs were concentrated and purified by ultracentrifugation combined by sucrose density gradient centrifugation, followed by verification by western blot, nanoparticle tracking analysis, and transmission electron microscopy. Protein composition in spermatozoa, SP and SPE, and protein difference in high and low motility SPEs were identified by LC-MS/MS proteomic analysis and functional analyzed by a comprehensive bioinformatic analysis.