Nanoparticles with an inner core of calcium phosphate, followed by layers of DNA and calcium phosphate and an outer layer of poly(ethyleneimine) (PEI) were prepared, characterized, and tested on different cell lines (HeLa, T24, and NIH3T3). A considerable increase in transfection efficiency was found for such nanoparticles, compared to the commercial reagent Polyfect(A (R)) (a cationic dendrimer). The DNA is incorporated into the nanoparticles and protected from the attack by enzymes (nucleases) inside the cytoplasm of cells. The outer layer of PEI leads to electrosteric colloidal stabilization and gives a positive charge to the nanoparticle, which is helpful for the penetration through the negatively charged cell membrane.