Interestingly, comparing to the chemical shift of the protons on the pyridinium unit, the signals of the protons on the azobenzene unit changed more obviously. The complexation-induced chemical shifts (Δδ) followed the order (see Supplementary Fig. S13): Hh > Hg > Hi > Hd > He = Hc > Hf > Hb > Ha. These shifts of methyl and both aromatic rings of the azobenzene unit were shielded by the pillararene, and the ring A is more shielded than others, which indicated that the aromatic rings of 2C-WP5A were located at the azobenzene unit of trans-G especially at ring A, instead of pyridinium unit. Although we failed to get the single crystal of the 2C-WP5Atrans-G, the 2D ROESY NMR experiment (see Supplementary Fig. S14) also supported our speculation of the binding mode. A 2D ROESY spectrum of an equimolar (5 mM) mixture of trans-G and 2C-WP5A in D2O was obtained to confirm the peak assignments. NOE correlation signals were observed between protons Hg, He and Hf on the azobenzene unit of trans-G and side chain protons H3 and phenyl moieties protons H2 on 2C-WP5A. The results showed that azobenzene unit was indeed threaded in the cavity of anionic 2C-WP5A, which was consistent with the above-mentioned 1H NMR results. To further confirm this result, we synthesized another guest molecule G′ with 1,4-diazabicyclo[2.2.2]octane group. The additional experiment results (see Supplementary Figs S15 and S16) also supported our conclusion that the azobenzene unit was positioned inside the cavity of 2C-WP5A. Recently, an inclusion complex of azo dye and cationic pillararenes was reported by Stoikov, which is also confirmed that azobenzene unit threaded in the cavity of pillararene45.