A cell penetrating peptide (KALA) and protamine sulfate (PS) were introduced to a nano-structured calcium carbonate based gene delivery system to obtain dual-functionalized KALA/PS/CaCO₃/DNA nanoparticles. The dual-functionalized nanoparticles were prepared by a facile co-precipitation method, during which all functional components were introduced to the nanoparticles simultaneously. For comparison, mono-functionalized gene delivery systems were also prepared to study the individual effects of PS and KALA. The size and zeta potential measurements indicated KALA/PS/CaCO₃/DNA nanoparticles exhibited a decreased size and an increased zeta potential. In vitro gene transfections mediated by different nanoparticles were carried out in 293T cells and HeLa cells. As compared with unmodified and mono-functionalized nanoparticles, dual-functionalized nanoparticles exhibited significantly improved gene delivery efficiency due to the enhanced cellular uptake and nuclear localization, especially at a low DNA concentration. The dual-functionalized nanoparticles have promising applications in gene therapy since the therapeutic nucleic acid reaching target cells is usually limited in the practical application.