We examined the inhibitory effect of cationic polyrotaxanes, which consist of alpha-cyclodextrins threaded on a poly(ethylene glycol) (PEG) chain, on the activity of the intestinal carnitine/organic cation transporter, OCTN2, in OCTN2 gene-transfected HEK293/PDZK1 cells. The cationic polyrotaxanes effectively inhibited the OCTN2-mediated carnitine transport. Polyrotaxanes with a longer PEG chain exhibited a greater inhibitory effect, possibly owing to multivalent interactions with binding sites on OCTN2. These cationic polyrotaxanes were far less cytotoxic than conventional polycations, and are therefore interesting candidates as low-toxicity inhibitors of cation transport at cell surfaces.