How do α-cyclodextrins self-Organize on a polymer chain? The relative conformation of the mobile cyclic molecules of a polyrotaxane has been analyzed quantitatively by means of both molecular dynamics and Monte Carlo simulations. Here, the polyrotaxane is formed by several α-cyclodextrins (α-CDs) threaded onto a poly(ethylene glycol) (PEG) chain. The dimerization free energies for three possible spatial arrangements of two consecutive α-CDs, viz., head to head (HH), head to tail (HT), and tail to tail (TT), were determined. The computed dimerization free energies were then introduced into the theoretical framework of a lattice model to predict the percentage of HH and TT motifs in all possible arrangements, employing Monte Carlo simulations. Our results show that this percentage fluctuates when the number of CDs is less than eight and rapidly tends toward 73% when the latter is greater than eight. This theoretical estimate, which is dominated by the dimerization free energy, agrees well with experiments. Deconvolution of the free-energy profiles indicates that dimerization is controlled primarily by the formation of hydrogen bonds between two consecutive α-CDs, hence rationalizing why HH is more favorable than the other two spatial arrangements. The proposed method combining free-energy calculations with a lattice chain model is envisioned to be applied to other 1D chemical or biological self-assembly phenomena to help dissect the mechanisms that underlie the formation of the supramolecular assembly and control the relative conformation of its constituent cyclic compounds. J. Phys. Chem. C 2012.