Cyclodextrin-modified polycations have been studied widely due to their low cytotoxicity, low immunogenicity and the ability to form inclusion complexes. However, the influence of CD modification on cellular uptake and transfection efficiency of polyplexes is still unclear. In this research, cyclodextrin-modified polyethylenimines (PEI-CD) with different CD-grafting levels were synthesized, which were named PEI-CD₁₅ and PEI-CD₄₁, respectively, according to the CD number per PEI chain. CD modification showed great influence on the DNA condensation ability of the polycation. PEI-CD₁₅ could protect DNA completely above N/P ratio of 2. The particle sizes of these polyplexes were about 120 nm. However, PEI-CD₄₁ could not protect DNA below N/P of 6, and PEI-CD₄₁/DNA polyplexes were larger than 1 μm, even at N/P ratio of 10. Therefore, this research was mainly focused on PEI-CD₁₅. It was interesting that the PEI-CD₁₅/DNA polyplexes at N/P ratio of 8 and 10 displayed excellent stability in physiological salt conditions, probably due to the hydration shell of CDs. The influence of CD modification on the cellular uptake and transfection efficiency of polyplexes depended on the type of the cells. Uptake inhibition experiments indicated that PEI/DNA polyplexes were internalized by HEK293T cells by both clathrin-mediated endocytosis and caveolae-mediated endocytosis. The route of caveolae-mediated endocytosis was significantly promoted after CD modification. So the cell uptake and transfection efficiency of PEI-CD₁₅/DNA polyplexes were significantly improved for HEK293T cells. However, the uptake and transfection efficiency of PEI-CD₁₅/DNA polyplexes in HepG2 cells was similar to that of PEI/DNA polyplexes, probably due to the lack of endogenous caveolins.