Abstract Diamine reagents have been used to functionalize a [2]rotaxane building block bearing an activated pentafluorophenyl ester stopper. Upon a first acylation, an intermediate host‐guest complex with a terminal amine function is obtained. Dissociation of the intermediate occurs in solution and acylation of the released axle generates a [2]rotaxane with an elongated axle subunit. In contrast, the corresponding [3]rotaxane can be obtained if the reaction conditions are appropriate to stabilize the inclusion complex of the mono‐amine intermediate and the pillar[5]arene. This is the case when the stopper exchange is performed under mechanochemical solvent‐free conditions. Alternatively, if the newly introduced terminal amide group is large enough to prevent the dissociation, the second acylation provides exclusively a [3]rotaxane. On the other hand, detailed conformational analysis has been also carried out by variable temperature NMR investigations. A complete understanding of the shuttling motions of the pillar[5]arene subunit along the axles of the rotaxanes reported therein has been achieved with the help of density functional theory calculations.