Salmonella enterica is a versatile live carrier for the presentation of recombinant vaccine antigens. Fusion proteins of a type III secretion system effector and heterologous vaccine antigens can be translocated by live attenuated Salmonella strains and mediate protective immunity against infections. Here we investigated the use expression cassettes for translocated fusion protein consisting of effector SseF and antigens of Listeria monocytogenes after stable integration into the Salmonella chromosome. The efficacy of chromosomal expression cassettes was compared to plasmid-based expression cassettes. Our data indicate that live Salmonella vaccines with chromosomal expression cassettes for translocated fusion proteins, although only present in single copy, efficiently stimulate immune responses.