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We investigated the use of two previously described attenuated strains of Salmonella enterica subspecies enterica serovar Choleraesuis (S. Choleraesuis), DeltaphoP and DeltarpoS, compared with the commercial attenuated SC-54 strain, as bactofection vehicles, to deliver an epitope model (3xFLAG) to the intestinal immune system. The gene encoding the epitope 3xFLAG was subcloned into the pCMVbetam2A mammalian expression vector (creating pCMV3xFLAGm2A) and introduced into S. Choleraesuis strains. The 3xFLAG epitope was expressed efficiently in murine macrophage J774A.1 cell cultures infected with Salmonella DeltaphoP and DeltarpoS vehicles but not with SC-54, as shown by gene-specific quantitative real-time reverse-transcriptase PCR. The stability of pCMV3xFLAGm2A in each strain was determined in vitro in the absence of antibiotic selection, and in vivo following oral immunisation of BALB/c mice. Administration of the DNA vaccine to mice led to the production of 3xFLAG-specific serum IgG and intestinal IgA antibody responses in DeltarpoS and SC-54, and spleen cell secretion of IFN-gamma following specific 3xFLAG stimulation in DeltaphoP. All together, these results indicate that DeltaphoP, DeltarpoS and SC-54 that expressed 3xFLAG from pCMV3xFLAGm2A elicited a different biased immune response, in which the T-helper-1-like cellular immune response was predominant in DeltaphoP, whilst IgA-related mucosal immunity predominated in DeltarpoS and SC-54. We conclude that DeltaphoP and DeltarpoS of S. Choleraesuis are new promising candidates as vaccine bactofection vectors.