Cationic polyethylenimine (PEI)-based nonviral gene carriers have been desirable to overcome the limitations of viral vectors in gene therapy. A range of PEI derivatives were designed, synthesized, and evaluated for nonviral delivery applications of plasmid DNA (pDNA). Linolenic acid, lauric acid, and oleic acid were covalently conjugated with low-molecular-weight PEI ( ∼ 1200 Da) via two different linkers, gallic acid (GA) and -hydroxybenzoic acid (PHPA), that allows a differential loading of lipids per modified amine (3 vs 1, respectively). H NMR spectrum confirmed the expected structure of the conjugates as well as the level of lipid substitution. SYBR Green binding assay performed to investigate the 50% binding concentration (BC) of lipophilic polymers to pDNA revealed increased BC with an increased level of lipid substitution. The particle analysis determined that GA- and PHPA-modified lipopolymers gave pDNA complexes with ∼300 and ∼100 nm in size, respectively. At the polymer/pDNA ratio of 5.0, the ζ-potentials of the complexes were negative (-6.55 to -10.6 mV) unlike the complexes with the native PEI (+11.2 mV). The transfection experiments indicated that the prepared lipopolymers showed higher transfection in attachment-dependent cells than in suspension cells based on the expression of the reporter green fluorescent protein (GFP) gene. When loaded with Cy3-labeled pDNA, the lipopolymers exhibited effective cellular uptake in attachment-dependent cells while the cellular uptake was limited in suspension cells. These results demonstrate the potential of lipid-conjugated PEI via GA and PHPA linkers, which are promising for the modification of anchorage-dependent cells.