We studied the physicochemical properties of some cationic lipophosphoramidates used as gene vectors in an attempt to better understand the link between the nature of the hydrophobic chain and both physico-chemical properties and transfection efficiency. These compounds have an arsonium head group and various chain lengths and unsaturation numbers. The synthesis of cationic phospholipids with oleic (Guenin et al., 2000 [1]; Floch et al., 2000 [2]) or linoleic (Fraix et al., 2011 [3]; Le Gall et al., 2010 [4]) chains has already been reported by our group and their efficiency as gene carriers has been demonstrated. Four new compounds were synthesized which incorporated either C14:0, C18:0, C20:4 or C20:5 chains. The membrane fluidity was studied by fluorescence anisotropy measurements. The fusion of liposomes and lipoplexes with membrane models was studied by Förster Resonant Energy Transfer. Finally, DNA condensation was studied and the lipoplexes were tested in vitro to quantify their transfection efficiency. From the results obtained on these cationic lipophosphoramidates series, we show that aliphatic chain length and unsaturation number have an important influence on liposomes physicochemical properties and transfection efficiency. However there is no direct link between fluidity and fusion efficiency or between fluidity and DNA condensation. Nevertheless, it seems that for best transfection efficiency the compounds need to combine the properties of fluidity, fusion efficiency and DNA condensation efficiency. This was the case for the C18:1 and C18:2 compounds.