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The aim of this study was to improve the gene delivery efficacy of chitosan oligomer polyplexes by introducing a trisaccharide branch that targets cell-surface lectins. For this purpose, chitosan oligomers were substituted by a trisaccharide with the N-acetylglucosamine residue at the free end, and the ability of the trisaccharide-substituted chitosan oligomers (TCO) polyplexes to transfect various cell lines in vitro and lung tissue after in vivo administration to mice was investigated. Live-cell confocal microscopy showed improved cellular uptake in HEK 293 cells (11-fold, p < 0.001) for the TCO polyplexes compared with the linear chitosan oligomers. Colloidal stability was also enhanced with the substituted form, which suggests that the trisaccharide branch stabilised the polyplexes by means of a steric stabilisation mechanism. Interestingly, gene expression levels in the human liver hepatocyte (HepG2) cells were 10-fold higher with the TCO polyplexes than those mediated by polyethyleneimine. A similar improvement was obtained in a human bronchial epithelial cell line (16HBE14o-). Transfection with the TCO was significantly inhibited (by 30-80%). for all the cell lines tested, in the presence of the free trisaccharide branch, confirming lectin-mediated uptake. Finally, in vivo studies showed that, 24 h after lung administration to mice, luciferase gene expression was 4-fold higher with the TCO than with the corresponding linear chitosan oligomers.