Cationic lipids can form stable complexes, so-called lipoplexes, with DNA through electrostatic interactions. Positively charged lipoplexes interact strongly with negatively charged cell membranes and are taken up by the cell through an endocytic pathway. We have previously designed a novel cationic lipid consisting of polyamidoamine dendron of third generation and two dodecyl chains, designated as DL-G3-2C12 and synthesized by repetition of exhaustive Michael addition with methyl acrylate using di-n-dodecylamine as the core material and subsequent exhaustive amidation with ethylenediamine. The DL-G3-2C12 gave lipoplexes which exhibit efficient transfection of cells. In this study, polyamidoamine dendron-bearing lipid with two octadecyl chains DL-G3-2C18 was additionally synthesized by the same method using di-n-octadecylamine as the core material. We compared abilities of these dendron-bearing lipids to form complexes with DNA and to transfect cells. The influence of the difference in alkyl chain length of these dendron-bearing lipids on their functions as non-viral gene vectors was investigated. The ability of these dendron-bearing lipids to form a lipoplex with plasmid DNA was investigated. The DL-G3-2C18 formed a lipoplex with plasmid DNA more slowly, but produced more condensed lipoplexes compaction of plasmid DNA, when compared with the case of DL-G3-2C12. Next, we investigated destabilization of these lipoplexes upon addition of heparin, which is a polysaccharide with negatively charged groups. We found that the DL-G3-2C18 lipoplex required more heparin than DL-G3-2C12 lipoplex to cause dissociation of plasmid DNA from the lipoplexes. This result indicates that hydrophobic interaction through the longer alkyl chains of DL-G3-2C18 might increase stability of the lipoplexes. Finally, we examined the transfection of CV1 cells using these lipoplexes. While the DL-G3-2C12 lipoplexes significantly decreased their transfection activity in the presence of serum, the DL-G3-2C18 lipoplexes exhibited much higher serum-resistance. These results indicate that hydrophobic interaction of alkyl chain moieties greatly contribute to stability and transfection activity of the lipoplexes of dendron-bearing cationic lipids.