Cationic lipid/co-lipid combinations have been extensively explored in gene delivery as alternatives to viral vectors. To be established as a gold standard of chemical vectors, considerable improvement in their transfection efficiency is however required. Herein, we report a simple procedure to synthesize new cationic and co-lipids for the DNA transfection of dendritic cells (DCs). Seven α-amino-lipophosphonates featuring two aza-heterocycles with protonable sites (imidazole or pyridine) were synthesized and used as co-lipids in liposomes with cationic lipid. For each liposome, the cationic lipid is either the imidazolium lipophosphoramidate (Lipid 2) or α-amino-lipophosphonate containing basic tertiary aliphatic amine in the polar head group (Lipid 3b). The cationic lipids either with new co-lipids or DOPE formed positively charged nano-sized stable liposomes that effectively interact with plasmide DNA (pDNA) to produce lipoplexes. Membrane fusion studies showed that α-amino-phosphonates featuring imidazole moiety in the polar head group exhibited higher fusion at pH 5.5 than pH 7.4. This study suggests that the best formulations for the transfection of DCs (based on the % transfected cells and the intensity of EGFP-based fluorescence) are Lipid 2 associated with either 3a, 3d or DOPE and the cationic lipid 3b formulated with 3a or DOPE as helper lipid. Furthermore, Lipid 3a could be used as an alternative to DOPE as a helper lipid. Overall, these results indicate that novel imidazole containing α-amino-phosphonates can serve as effective transfection agents for the DC-based vaccines.