T-shaped spermine-based cationic lipids with identical and nonidentical hydrophobic tails having variable carbon lengths (from C10 to C18) were designed and synthesized. These lipids were characterized, and their structure-activity relationships were determined for DNA binding and transfection ability of these compounds when formulated as cationic liposomes. These liposomes were then applied as non-viral vectors to transfect HEK293T, HeLa, PC3, H460, HepG2, and Calu'3 cell lines with plasmid DNA encoding the green fluorescent protein. ST9, ST12 and ST13 with nonidentical tails could deliver DNA into HEK293T cells up to 60% under serum-free conditions. The lipid ST15 bearing nonidentical tails was found to be a potent gene transfer agent under 40% serum conditions in HEK293T and HeLa cells. Besides their low cytotoxicity, these lipoplexes also exhibited greater transfection efficiency than the commercially available transfection agent, Lipofectamine 3000.