Optimal DNA vaccine efficacy requires circumventing several obstacles, including low immunogenicity, a need for adjuvant, and the costs of purifying injection grade plasmid DNA. Bacterial delivery of plasmid DNA may provide an efficient and low-cost alternative to plasmid purification and injection. Also, the bacterial vector may exhibit potential as an immune adjuvant in vivo. Thus, we elected to examine the use of cell-wall-deficient Listeria monocytogenes as a DNA delivery vehicle in vitro. First, the D-alanine-deficient (Delta dal-dat) L. monocytogenes strain DP-L3506, which undergoes autolysis inside eukaryotic host cells in the absence of D-alanine, was transformed with a plasmid encoding green fluorescent protein (GFP) under control of the CMV promoter (pAM-EGFP). Then COS-7 and MC57G cell lines were infected with the transformed DP-L3506 at various multiplicities of infection (MOI) in the presence or absence of D-alanine. Subsequent GFP expression was observed in both cell lines by 24 h post-infection with DP-L3506(pAM- EGFP). Notably, no GFP positive cells were observed when D-alanine was omitted. Although transfection efficiency initially increased as a result of D-alanine supplementation, high concentration or long-term supplementation led to sustained bacterial growth that killed the infected host cells, resulting in fewer GFP-expressing cells. Thus, efficient DNA delivery by transformed bacteria must balance bacterial invasion and survival with target cell health and survival.