Updated project metadata. Seminal plasma contains a large number of extracellular vesicles (EVs). However, the roles of these EVs and their interactions with sperm are not clear. To identify the important molecules affecting sperm motility in EVs, we performed proteomics analysis of SPEVs from the two groups with different sperm motilities. In total, 1435 exosomal proteins covered a broad range of protein classes, such as enzymes, cytoskeletal proteins, extracellular proteins and transcription factors. We identified 76 DEPs (differentially expressed protein) from the SPEVs of boars with high- and low-sperm motility phenotypes (|FC| ≥1.5, P≤ 0.05). A total of 38 proteins were downregulated in the L group compared with the H group, while 6 proteins were significantly upregulated. In addition, 28 proteins were identified only in the H group, and 4 proteins were identified only in the L group. The GO and KEGG analyses showed that the DEPs were associated primarily with negative regulation of cell motility, extracellular exosomes, the extracellular space, cell-cell adhesion and phagosome biological activities. The present fndings represent a major and novel contribution to the study of boar seminal extracellular vesicles proteins.