A series of linear polyester-based, α-cyclodextrin (CD)-threaded polyrotaxanes (PRTx) were synthesized for siRNA delivery. The investigation into the effect of the presence of polyester linkages in polyrotaxane established the structural–activity relationship between polyrotaxane and siRNA transfection efficiency. The ester-based polyrotaxane exhibited higher threading efficiency than poly­(ethylene glycol)-based polyrotaxane. The threading efficiency is the driving force for transfection, as it regulates the positive charge density on polyrotaxane. Polyester-based polyrotaxane formed stable and effective transfection nanoplexes with siRNA at lower N/P ratios, signifying the high gene loading capacity of the developed supramolecular vectors. Our findings suggest that biochemical properties of the transfection complexes depend on the structure of the axis and threading efficiency of polyrotaxane, which further influences the transfection efficiency. The enhanced gene silencing efficiency and safety are compared with those of extensively explored poly­(ethylene glycol)-based polyrotaxane, polyethyleneimine (a gold standard), and lipofectamine (a commercial standard), which are used as siRNA delivery vectors.