Lipid-mediated delivery of DNA into cells holds great promise both for gene therapy and basic research applications. The primary approach to improving transfection efficiency is the design and synthesis of novel cationic lipids. Alternatively, using the synergistic effect of different cationic mixtures can provide another approach to increasing transfection efficiency. This paper describes the synergistic effect of lipids with different polarheads, central core structures and hydrophobic tails. The enhancement of cellular transfection into HEK293 cells was observed by combining two lipids having aminoglycerol and di(hydroxylethyl)amino core structures at a 1 : 1 weight ratio. Additionally, the liposome formation of these lipids with the helper lipid, 1,2-dioleoyl-propyl-3-phosphatidylethanolamine (DOPE), at the weight ratio of 1 : 1 can provide higher transfection efficiency into HEK293, MCF-7 and HeLa cells than Lipofectamine™ 2000. Our finding indicated that cationic liposomes comprised of a mixture of lipids with different polarheads, central core structures and hydrophobic tails should be very promising in liposome-mediated gene delivery in vitro and in vivo .