Listeria monocytogenes is a facultative intracellular bacterium that enters a variety of non-professional mammalian cells by triggered phagocytosis ("zipper mechanism") and replicates in the cytosol of the infected host cells. Therefore, it is a promising vaccine vector for the presentation of passenger antigens to the MHC class II and especially class I pathways. Here, we review recent progress made in our laboratory on the development of novel attenuated L. monocytogenes carrier strains for the delivery of heterologous antigens or antigen-encoding DNA and RNA to eukaryotic host cells. Based on the deletion of the chromosomal copy of the tryptophanyl-tRNA synthetase gene (trpS) and plasmid-based in trans complementation of the same, we were able to establish a balanced-lethal plasmid system in L. monocytogenes. Safety concerns in the antigen delivery in vivo were addressed by chromosomal deletion of genes in the basic branch of the aromatic amino acid pathway, resulting in safe, attenuated L. monocytogenes carrier strains. Furthermore, plasmid-based expression of a cytosolically expressed phage lysin resulted in a self-destructing carrier strain that has been successfully used for the delivery of antigens as well as antigen-encoding plasmid DNA and particularly mRNA, therefore overcoming bottlenecks that have been shown to exist for bacteria-mediated DNA delivery.