Mycobacterium avium subsp. paratuberculosis (MAP) parasitizes macrophages of ruminant animals, ensuring its survival by blocking phagosome maturation and the induction of antimicrobial defences. Numerous secreted proteins have been implicated in subverting macrophage defences, however the mechanisms transporting these components are undefined. This research investigated the composition of MAP membrane vesicles (MVs) and identified MV protein composition, and antigenic MV proteins. Transmission electron microscopy visualized membrane vesicles that ranged from 20 to 270 nm in size, and immunoblotting with serum antibodies from MAP infected cattle identified seven antigenic proteins including previously identified secreted virulence factors of MAP. Collectively this work showed MVs of MAP contain proteins derived from the cell membrane, cytosol, and secreted proteins. As MAP MVs are secreted and can interact with host immune response cells, they may of be value in the search for MAP-specific epitopes for use in the detection of animals sub-clinically infected with MAP.