New amphiphilic pyridine derivatives containing dodecyloxycarbonyl substituents at positions 3 and 5 and cationic moieties at positions 2 and 6 have been designed and synthesised. Compounds of this type can be considered as synthetic lipids. The corresponding 1,4-dihydropyridine (1,4-DHP) derivatives have earlier been proposed as a promising tool for plasmid DNA (pDNA) delivery in vitro. In this work studies of the self-assembling properties of amphiphilic pyridine derivatives leading to the formation of liposomes, determination of particle size, zeta-potential and critical micelle concentration (CMC) with dynamic light scattering (DLS) measurements are described. Furthermore, thermal analysis of pyridine derivatives was performed using thermogravimetry analysis (TGA) and differential thermal analysis (DTA) as well as the ability to deliver the pEGFP-C1 plasmid DNA (that encodes GFP reporter) into the Baby hamster kidney-derived (BHK-21) cell line was used for evaluation of gene delivery properties. We have revealed that the new pyridine derivatives possessed self-assembling properties which were proved by formation of nanoparticles with the average size from 115 to 743 nm, the zeta-potentials in the range of 48–79 mV and CMC values in the range of 2–67 μM. DTA data showed that all processes were endothermic for all compounds. Additionally, we established that among the tested pyridines the representatives with N-methylpyrrolidinium or pyridinium moieties as cationic head-group at the positions 2 and 6 possessed higher pEGFP-C1 transfection activity into the BHK-21 cell line. Nevertheless, the obtained results indicated that correlation of the physicochemical, structural properties and gene delivery activities of the tested compounds were not completely elucidated yet. On the other hand, the synthesised pyridines as possible metabolites of promising delivery systems on the 1,4-DHP core possessed lower pDNA transfection activity than the corresponding 1,4-DHP amphiphiles.