The host–guest interactions of cationic (AcH⁺) and neutral (Ac) forms of the dye acridine with the macrocyclic host p-sulfonatocalix[6]arene (SCX6) were investigated by using ground-state absorption, steady-state and time-resolved fluorescence, and NMR measurements. The cationic form undergoes significant complexation with SCX6 (K_eq=2.5dx10⁴m^-1), causing a sharp decrease in the fluorescence intensity and severe quenching in the excited-state lifetime of the dye. The strong binding of the AcH⁺ form of the dye with SCX6 is attributed to ion–ion interactions involving the sulfonato groups (SO₃^-) of SCX6 and the positively charged AcH⁺ at pH of approximately 4.3. Whereas, the neutral Ac form of the dye undergoes weak complexation with SCX6 (K_eq=0.9x10^3_M-1) and the binding constant is lowered by one order of magnitude compared with that of the SCX6–AcH⁺ system. The strong affinity of SCX6 to the protonated form leads to a large upward pKa shift (≈2 units) in the dye. In contrast, strong emission quenching upon SCX6 interaction and the regeneration of fluorescence intensity of the dye in the presence of Gd³⁺ through competitive binding have also been demonstrated.