The structure of the cationic moiety of amphiphiles is a key factor which directly influences their transfection efficacy. Accordingly, in the present work, we have synthesized three new lipophosphoramide-based amphiphilic compounds incorporating a methoxy 5, hydroxyl 6, or dihydroxyl 7 functional group in their cationic part. Gene delivery efficacies of these novel vectors were compared to our benchmark compound, the arsenolipophosphoramidate KLN47, and to its trimethylammonium (TMA) analogue 4. We next studied the characteristics (size, ζ potential) of the nanometric assemblies formed (liposomes and lipid/DNA complexes), and the DNA binding ability of the cationic liposomes was characterized at the physicochemical level. In vitro, all of the cationic lipids evaluated were efficient not only to condense plasmids but also to transfect two types of human airway epithelial cells. Interestingly, in vivo administration to mice (via simple tail vein injection) showed that compound 6 was the most efficient in transfecting the lungs when compared to that of the other cationic lipids studied, including compound KLN47. All of these results suggest that a hydroxyethyldimethylammonium (HE-DMA) polar head could be a valuable alternative to a trimethylarsonium (TMAs) polar head and that they also invite further evaluation of the in vivo potential of compound 6 using more clinically relevant delivery procedures.