To avoid adverse immunogenic response invoked by viral vectors, increasing non-viral alternatives including peptide-based carriers have been proposed. In this paper, cationic and amphiphilic cholesterol conjugated oligopeptides containing a histidine block (5 or 10 residues) and an arginine block (10 residues), i.e. Chol-H5R10 and Chol-H10R10 were designed and synthesized. They were self-assembled into cationic core/shell nanoparticles in aqueous solution at concentrations of 17.8 and 28.2 mg/L respectively. These cationic nanoparticles condensed DNA efficiently. After being complexed with DNA, the particles had small average size of about 200 nm and high zeta potential of about 60 mV at higher N/P ratios (molar ratio of histidine and arginine content in peptide to phosphorus content in DNA). The nanoparticles induced much higher luciferase expression level in both human embryonic kidney HEK293 and human liver carcinoma HepG2 cell lines than the peptides without cholesterol because the formation of nanoparticles increased the local density of cationic charge, leading to more efficient DNA binding and higher zeta potential of DNA complexes. The luciferase expression level mediated by Chol-H10R10 nanoparticles was almost 10 times higher than that induced by Chol-H5R10 nanoparticles probably because an increased length of histidine led to higher capacity of endosomal escape and stronger DNA binding ability. The luciferase expression level induced by Chol-H10R10 nanoparticles was comparable to that mediated by PEI at the optimal N/P ratio (i.e. 10), but the cytotoxicity of nanoparticles was lower. In conclusion, Chol-H10R10 nanoparticles may be a promising gene delivery carrier.