Transient expression systems for plants have several advantages for the development of innovative plant-made vaccines. This technology has the potential to render veterinary vaccines against relevant diseases, such as paratuberculosis, a chronic intestinal infectious disease affecting livestock. This study showed that Agrobacterium -mediated transient transformation of maize seedlings allows the production of the Ag85B antigen from Mycobacterium avium subsp. paratuberculosis (MAP) at levels up to 11.35 µg g −1 dry weight. Moreover, BALB/c mice orally and subcutaneously immunized with the maize-made Ag85B vaccine produced specific IgG and IgA antibodies. Therefore, this platform is proposed as a rapid approach to produce the functional MAPAg85B antigen at sufficient yields to induce specific humoral immune responses in animal testing, which constitutes the first step in the development of a plant-made oral vaccine against paratuberculosis. Key message This paper describes for the first time, the transient transformation of maize seedlings to produce Ag85B vaccine from Mycobacterium avium subsp. paratuberculosis . The plant-produced antigen showed strong immunoreactivity in immunized mice.