Polyethylenimine (PEI), especially PEI 25 kDa, has been widely studied for deliveryof nucleic acid drugs both in vitro and in vivo. However, it lacks degradable linkages and istoo toxic for therapeutic applications. Hence, low-molecular-weight PEI has been explored asan alternative to PEI 25 kDa. To reduce cytotoxicity and increase transfection efficiency, wedesigned and synthesized a novel small-molecular-weight PEI derivative (PEI-Et, Mn: 1220,Mw: 2895) with ethylene biscarbamate linkages. PEI-Et carried the ability to condense plasmidDNA (pDNA) into nanoparticles. Gel retardation assay showed complete condensationof pDNA at w/w ratios that exceeded three. The particle size of polymer/pDNA complexeswas between 130 nm and 180 nm and zeta potential was 5–10 mV, which were appropriatefor cell endocytosis. The morphology of PEI-Et/pDNA complexes observed by atomic forcemicroscopy (AFM) was spherically shaped with diameters of 110–190 nm. The transfectionefficiency of polymer/pDNA complexes as determined with the luciferase activity assay as wellas fluorescence-activated cell-sorting analysis (FACS) was higher than commercially availablePEI 25 kDa and Lipofectamine 2000 in various cell lines. Also, the polymer exhibited significantlylower cytotoxicity compared to PEI 25 kDa at the same concentration in three cell lines.Therefore, our results indicated that the PEI-Et would be a promising candidate for safe andefficient gene delivery in gene therapy.