Rotavirus is the main cause of gastroenteritis in children worldwide, and the World Health Organisation has recommended that a rotavirus vaccine should be included in all infant immunization programmes. VP6 is the most immunogenic rotavirus subunit and is a potential target for an oral subunit vaccine. VP6 accumulated at up to 3% of total soluble protein in the young leaves of transplastomic tobacco plants, but the protein was unstable and was lost as the leaves aged. The aim of this study was to alter the 5′-untranslated region (5′-UTR) and the 5′ end of the coding region of VP6 cDNA in an attempt to increase the expression and stability of VP6 protein in tobacco chloroplasts. The inclusion of the 5′-UTR from gene 10 of bacteriophage T7 (T7g10) and the addition of 15 nucleotides, encoding five additional amino acid residues, at the 5′ end of the coding region increased the expression to >15% of total leaf protein and stabilized the protein in ageing leaves. Plants containing VP6 expression constructs with the rbcL 5′-UTR and with the native VP6 5′ end of the coding region produced VP6 protein at only 1.9% of total leaf protein. Both the T7g10 5′-UTR and the additional 15 nucleotides increased transcript accumulation and translational efficiency compared with VP6 constructs containing the rbcL 5′-UTR. The VP6 protein produced from all gene constructs appeared to be susceptible to proteolytic processing at its N-terminal region. However, in all transplastomic lines, VP6 proteins assembled into the trimeric form found in the rotavirus capsid.