The most definite feature in polyrotaxanes, in which many cyclic compounds are threaded onto a linear polymeric chains capped with bulky end-groups, is the mobility of cyclic compounds: these cyclic compounds may rotate and/or slide along the polymeric chain. Our previous studies have clarified that the mobility of ligands linked to the cyclic compounds is closely related to enhancing multivalent interaction with biological systems. This concept is now exploiting more practical applications for drug delivery such as gene delivery. We have designed biocleavable polyrotaxanes that have a necklace-like structure between many dimethylaminoethylcarbamoyl-α-cyclodextrins (DMAE-α-CDs) and a disulfide (SS)-introduced poly(ethylene glycol) (PEG) chain. The polyrotaxanes were found to show sufficient cleavage of S-S linkages under reducible condition, which led to triggering pDNA release via the dissociation of the non-covalent linkages between DMAE-α-CDs and the PEG chain. The polyrotaxanes were finally clarified to exhibit great transfection activity as well as non cytotoxicity.