Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG−bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI−PEG−PEI triblock copolymers with controlled compositions. Two copolymers, PEI−PEG−PEI 2100−3400−2100 and 4000−3400−4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate ζ-potentials (+10 mV) at polymer/plasmid weight ratios ≥1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI−PEG−PEI 4000−3400−4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI−PEG−PEI 4000−3400−4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI−PEG−PEI polyplexes. These PEI−PEG−PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI−PEG−PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.