Cationic microparticles for DNA adsorption were formulated by blending poly(lactide-co-glycolide) (PLGA) (50:50), with different cationic agents, either PEI 25 kDa (polyethylenimine) or CTAB (cetyl-trimethyl-ammonium-bromide). The aim was to create adjuvant delivery systems increasing the efficiency of DNA vaccines. Microparticles formulated with 10% PEI exhibited a highly positive zeta-potential, small particle sizes, in contrast to particles prepared with CTAB, which revealed highly aggregated structures in scanning electron micrographs. PEI 10% microparticles efficiently adsorbed DNA and protected DNA from enzymatic degradation. Microparticles with up to 10% PEI did not affect membrane integrity whereas CTAB particles showed higher LDH release. Transfection efficiencies were assessed using a luciferase reporter gene assay compared to naked DNA and PEI/DNA polyplexes. DNA adsorbed onto microspheres with 10% or 50% PEI generally had higher transfection efficiencies than CTAB but reached lower expression levels than PEI/DNA polyplexes alone. This documented the intact release of DNA. The mechanism of gene delivery to non-phagocytic cells was studied via covalent fluorescence labeling of both the DNA and PEI by confocal microscopy and suggested uptake of DNA. Immunization of mice was performed using plasmids encoding immunodominant antigens of Listeria monocytogenes adsorbed onto RG 502 H+PEI 10% microparticles. The efficiency was tested by intravenous challenge with an otherwise lethal dose of L. monocytogenes. PLGA+PEI microspheres can be used as adjuvant delivery systems for DNA but further optimization is necessary to exploit their full potential.