Ternary nanoparticles, protamine sulfate-calcium carbonate-plasmid DNA (PS-CaCO3-DNA), were prepared for efficient gene delivery. By adding the cationic polypeptide PS in the co-precipitation system of calcium carbonate and DNA, PS-CaCO3-DNA nanoparticles could be formed by self-assembly facilely. The effect of PS on the properties of the ternary nanoparticles was studied by varying the PS amount in the nanoparticles. The size and ζ-potential measurements indicated that the ternary nanoparticles with an appropriate PS amount exhibited a decreased size and an increased ζ-potential. The in vitro gene transfections mediated by different nanoparticles in 293T cells and HeLa cells were carried out in the presence of 10% fetal bovine serum, using pGL3-Luc and pEGFP-C1 as reporter plasmids. As compared with both PS-DNA nanoparticles and CaCO3-DNA nanoparticles, PS-CaCO3-DNA nanoparticles exhibited significantly enhanced gene delivery efficiency, which was higher than that of Lipofectamine 2000-DNA. Confocal microscopy observation showed that PS-CaCO3-DNA nanoparticles could efficiently deliver DNA to cell nuclei. These results indicated that the ternary PS-CaCO3-DNA nanoparticles prepared in this study have promising applications in gene delivery.