Vaccines can reduce the use of antibiotics by preventing specific infective diseases in pigs. Plant-based edible vaccines are particularly attractive because, upon oral ingestion via feed, they can elicit the local immune system against a foreign disease-causing organism. The aim of this study was to engineer two different independent lines of tobacco plants for the seed-specific expression of immunogenic proteins of VTEC as a model of an edible vaccine. For each antigen, fifty Nicotiana tabacum L. cv Xanthi leaf disks were transformed by agroinfection for the seed-specific expression of the structural parts of the fimbrial subunit FedF of F18 and the B-subunit of Vt2e genes. The synthetic genes, optimized by the codon adaptation index for their expression in tobacco, were inserted into expression cassettes under the control of β-conglycinin promoter. Regenerated tobacco plants (T0) were characterized by molecular and immunoenzymatic techniques. Our results showed that both FedF and Vt2eB genes were integrated into tobacco genome efficiently (> 80%) and they are also maintained in the second generation (T1). Western blotting analyses carried out on the positive producing lines, showed the tissue-specific expression in seeds and the temporal protein accumulation in the mid-late maturation phases. The enzyme-linked immunosorbent assay showed seed expression levels of 0.09 to 0.29% (from 138 to 444 µg/g of seeds) and 0.21 to 0.43% (from 321 to 658 µg/g of seeds) of total soluble protein for the FedF and Vt2eB antigens, respectively. This study confirmed the seed-specific expression of the selected antigens in plant seeds. The expression level is suitable for seed-based edible vaccination systems, which could represent a cost-effective way to prevent VTEC infection. Our findings encourage further in vivo studies focused on the activation of the local immune response.