A DNA construct containing the cholera toxin B subunit (CTB) gene genetically fused to a nucleotide sequence encoding three copies of tandemly repeated diabetes-associated autoantigen, the B chain of human insulin, was produced and transferred into low-nicotine tobaccos by Agrobacterium. Integration of the fusion gene into the plant genome was confirmed by polymerase chain reaction (PCR). The results of immunoblot analysis verified the synthesis and assembly of the fusion protein into pentamers in transgenic tobacco. GM1?ELISA showed that the plant-derived fusion protein retained GM1?ganglioside receptor binding specificity. The fusion protein accounted for 0.11% of the total leaf protein. The production of transgenic plants expressing CTB?InsB3 offers a new opportunity to test plant-based oral antigen therapy against autoimmune diabetes by inducing oral tolerance.