In this study we synthesized a pair of orientationally isomeric [2]rotaxanes (1 and 2) featuring an α-cyclodextrin (α-CD) as the macrocyclic component and phenylurea and 2-hydroxybenzophenone moieties as stoppers of the axle-like component. The presence of the α-CD component enhanced the anion recognition ability of the urea unit in isomer 1, with the OH groups on the wide rim of α-CD undergoing hydrogen bonding with the anions. The association constant for the interaction between 1 and the acetate (AcO⁻) anion was more than four times that of the corresponding non-interlocked dumbbell-shaped molecule and the AcO⁻ anion. The other isomer 2, in which the wide rim of the α-CD component faced the 2-hydroxybenzophenone unit, was preferentially deprotonated by the AcO⁻ anion to give the phenoxide anion, which was stabilized through noncovalent interactions with the OH groups on the wide rim of the α-CD unit in an organic solvent; in contrast, the AcO⁻ anion did not deprotonate the corresponding non-interlocked dumbbell-shaped molecule.