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Cationic liposomes (CLs) composed of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposomes) have been classified as one of the most efficient gene delivery systems. Our study aims to examine the effect of the molar ratio of DC-Chol/DOPE, PEGylation and serum on the pDNA (plasmid pDNA) and siRNA (small interfering RNA) transfection of DC-Chol/DOPE liposomes. The results showed that the most efficient DC-Chol/DOPE liposomes for pDNA or siRNA delivery were at a 1:2 or 1:1 molar ratio of DC-Chol/DOPE, respectively. The transfection efficiency of DC-Chol/DOPE liposomes increased along with increased weight ratio of DC-Chol/siRNA. However, the pDNA transfection efficiency decreased along with increased weight ratio of DC-Chol/pDNA from 3/1. As expected, PEGylation decreased siRNA and pDNA transfection efficiency of DC-Chol/DOPE liposomes. In PEGylated DC-Chol/DOPE liposomes, increased weight ratio of DC-Chol/pDNA from 3/1 did not lead to higher pDNA transfection efficiency, whereas increased weight ratio of DC-Chol/siRNA resulted in increased siRNA transfection efficiency. Furthermore, the serum did not significantly inhibit the pDNA and siRNA transfection efficiency of DC-Chol/DOPE liposomes. In conclusion, our results elucidated the influence factors of DC-Chol/DOPE liposome transfection and would reveal that siRNA and pDNA transfection mechanisms were different in DC-Chol/DOPE liposomes.