The clarification of the structure–activity relationships of non-viral gene delivery vectors is of great importance. A series of novel cyclen-based cationic lipids were synthesized and applied as gene carriers. The structures of these fifteen lipids varied with different linking groups and hydrophobic tails. The liposomes formed by these lipids and helper lipid DOPE could efficiently bind DNA and condense it into nano-sized particles with positive zeta-potentials and protect DNA from enzymatic digestion. Results revealed that the hydroxyl group or aromatic ring in the lipid structure would affect their DNA binding and transfection ability. Lipids with double hydrophobic tails gave much higher transfection efficiencies than those with a single tail, and aromatic rings in the lipid backbone might facilitate the transfection. The transfection efficiency could be further improved by optimizing the hydrophobic tails. The lipid with C14 tails gave a much higher efficiency than its analogs. A cytotoxicity assay showed that the lipids generally gave higher cell viabilities than a commercially available transfection reagent. It was suggested that such cationic lipids may act as promising non-viral gene delivery carriers.