Abstract Bovine respiratory syncytial virus (BSRV) is a major cause of respiratory disease in calves and is closely related to human RSV. Bovine viral diarrhoea virus (BVDV) is responsible for disease that affects the respiratory, reproductive, immune and enteric systems of cattle. These diseases are a huge problem globally for the livestock industry and a mucosally targeted, bivalent vaccine which prevents infection and spread of these viruses would be valuable. We investigated the influence of the site of expression of the BVDV E2 protein on induction of immune responses using recombinant (r)BRSV expressing wild-type and mutant forms of the E2 protein targeted to different sites within polarised epithelial cells. Calves vaccinated intranasally with rBRSV expressing the E2 protein at the basolateral surface induced higher levels of E2-specific serum antibodies than animals vaccinated with rBRSV expressing E2 at the apical membrane, intracellularly or when secreted from cells. Targeting E2 to the basolateral surface also induced E2-specific IgA antibodies in respiratory secretions without compromising RSV-specific responses. Calves vaccinated intranasally with rBRSV expressing E2 targeted to the basolateral surface were completely protected against challenge with BRSV, and were also protected against nasopharyngeal excretion of BVDV. These studies highlight the importance of the site of expression of foreign proteins in polarised epithelial cells on priming of immune responses.