A new series of cationic block copolymers PHML-b-PMAGal and statistical copolymers P(HML-st-MAGal) with pendant natural galactose and (l-)-lysine moieties were prepared via RAFT (reversible addition-fragmentation chain-transfer) polymerization. The block/statistical copolymers showed a high plasmid DNA binding affinity (N/P < 2) and the as-formed polyplexes were spherical nanoparticles with the average size of 100-300 nm and surface zeta potentials of +30.2 to +46.3 mV. The cytotoxicity and gene transfection efficacy of the PHML-b-PMAGal and P(HML-st-MAGal) vectors strongly depend on the polymer architectures (block/statistical) and the galactose content. Notably, the statistical copolymer P(HML40-st-MAGal4) with 4.8% galactose content showed the highest gene transfection efficiency among the synthesized cationic polymers, 6.8-fold higher than that of the "gold standard" bPEI-25k in the presence of 10% FBS (fetal bovine serum) in various cell lines. An intracellular uptake mechanism (with 10% FBS) study demonstrated that the P(HML40-st-MAGal4)/pDNA polyplexes entered H1299 cells mainly through caveolae-mediated endocytosis and microtubule-dependent endocytosis pathways. Moreover, the fluorescence imaging study showed that the P(HML40-st-MAGal4)/pDNA polyplexes possessed an obvious "lysosomal escaping" effect that led to efficient pDNA release, which might interpret the fact of the significant increase of the related gene transfection efficiency. Moreover, it could be anticipated that the P(HML40-st-MAGal4) cationic glycopolymer might be employed as a low toxic, highly efficient and serum-compatible gene carrier for practical applications.