The success in gene therapy relies strongly on new efficient gene delivery vectors. Non-viral vectors are highly attractive, since they can deliver large quantities of genetic information and are low immunogenic. In this respect, unsymmetrical bolaamphiphiles (bolas) bearing two different hydrophilic head-groups connected by a hydrophobic spacer could be an attractive alternative for vector design, as they can form well-defined supramolecular nanostructures (nanovesicles and nanotubes) that are more stable than those formed with lipids. The aim of the present work was to develop new gene delivery vectors based on bolas capable to form such nanostructures hosting a DNA molecule and exposing at their external surface neutral ligands. For this purpose, a variety of bolas, bearing cationic and neutral groups connected by a hydrophobic spacer, were obtained by multi-step organic synthesis. Their characterization by different instrumental techniques suggested that the nature of the head groups as well as the hydrophobic spacer defines the bola self-assembly, their interaction with DNA and the morphology of their complexes (bolaplexes). While the first two generations of bolas lack the essential features of nonviral vectors, the final third generation was found highly promising. The latter showed a strong interaction with DNA, the formation of small bolaplexes and good transfection efficiency in different cell lines. The increase in transfection efficiency in presence of DOPE or chloroquine suggested that the key barrier for their internalization could be the lipid fusion and the endosomal escape. Some bolaplex formulations showed a transfection efficiency comparable to the best commercial transfection agents. Thus, the present work introduces a new class of efficient transfection agents based on unsymmetrical bolaamphiphiles.