A series of monosubstituted pillar[5]arenes (PIn) bearing a fluorescent probe (E)-4-[4-(dimethylamino)styryl-]-1-pyridinium were synthesized and characterized. The conformations of the monosubstituted pillar[5]arenes were investigated systematically by NMR and fluorescence spectroscopy and were found to be dependent on solvent polarity, concentration, temperature and linker length. PI1 with a short linker remained uncomplexed in DMSO, whereas it formed a polymer at high concentration in chloroform. As the linker length increased, PI2-4 could exist in equilibrium between self-inclusion monomer and intermolecular complexes. The increase of concentration led to the formation of a polymer in DMSO and a self-penetrating dimer in chloroform. The results provided information on the modulation of supramolecular assemblies by various factors.