Cationic glycopolymers have shown to be excellent candidates for the fabrication of gene delivery devices due to their ability to electrostatically interact with negatively charged nucleic acids and the carbohydrate residues ensure enhanced stability and low toxicity of the polyplexes. The ability to engineer the polymers for optimized compositions, molecular weights, and architectures is critical in the design of effective gene delivery vehicles. Therefore, in this study, the aqueous reversible addition-fragmentation chain transfer polymerization (RAFT) was used to synthesize well-defined cationic glycopolymers with various cationic segments. For the preparation of cationic parts, N-[3-(dimethylamino)propyl]methacrylamide hydrochloride (DMAPMA·HCl), water-soluble methacrylamide monomer containing tertiary amine, was polymerized to produce DMAPMA·HCl homopolymer, which was then used as macroCTA in the block copolymerization with two other methacrylamide monomers containing different pendant groups, namely, 2-aminoethyl methacrylamide hydrochloride (AEMA) (with primary amine) and N-(3-aminopropyl) morpholine methacrylamide (MPMA) (with morpholine ring). In addition, statistical copolymers of DMAPMA.HCl with either AEMA or MPMA were also synthesized. All resulting cationic polymers were utilized as macroCTA for the RAFT copolymerization with 2-lactobionamidoethyl methacrylamide (LAEMA), which consists of the pendent galactose residues to achieve DMAPMA·HCl-based glycopolymers. From the in vitro cytotoxicity study, the cationic glycopolymers showed better cell viabilities than the corresponding cationic homopolymers. Furthermore, complexation of the cationic polymers with siRNA, cellular uptake of the resulting polyplexes, and gene knockdown efficiencies were evaluated. All cationic polymers/glycopolymers demonstrated good complexation ability with siRNA at low weight ratios. Among these cationic polymer-siRNA polyplexes, the polyplexes prepared from the two glycopolymers, P(DMAPMA65-b-LAEMA15) and P[(DMAPMA65-b-MPMA63)-b-LAEMA16], showed outstanding results in the cellular uptake, high EGFR knockdown, and low post-transfection toxicity, suggesting the great potential in siRNA delivery of these novel glycopolymers.