For the development of polyplex systems showing a high transfection efficacy without a large excess of polycations, a lysine (Lys) unit as a DNA anchoring moiety was introduced into the amino acid sequence in poly(ethylene glycol)-b-cationic poly(N-substituted asparagine) with a flanking N-(2-aminoethyl)-2-aminoethyl group (PEG-b-Asp(DET)) resulting in PEG-b-P[Lys/Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. PEG-b-P[Lys/Asp(DET)]/DNA polyplexes exhibited a narrow size distribution of approximately 90 nm without secondary aggregates at the stoichiometric N/P 1, suggesting the formation of PEG-shielded polyplex micelles. The introduction of Lys units into the catiomer sequence facilitated cellular uptake and a 100-fold higher level of gene expression with PEG-b-P[Lys/Asp(DET)]/DNA polyplex micelles prepared even at a lowered N/P 2, possibly due to the enhanced association power of the anchoring Lys units.