• Protonated (AOH + ) and neutral (AO) forms of the acridine orange dye showed contrasting changes in their photophysical properties on complexation with γCD. • In the AOH + - γCD system, both dimeric AOH + -γCD and monomeric AOH + -γCD complexes are formed. • In the AO-γCD system, mainly the dimeric AO-γCD complexes are formed. • Nature of the protonation state of the dye and involvement of mainly hydrophobic interactions play a governing role in the present systems. Current study explores the contrasting supramolecular recognition behavior of protonated (AOH + , pH ∼ 7) and neutral (AO, pH ∼ 12) forms of the acridine orange dye with γ-cyclodextrin (γCD) host, investigated using various photophysical and NMR measurements. Intriguingly, AOH + undergoes sequential multi-mode binding interactions with the γCD host showing the predominant formation of dimeric AOH + -γCD inclusion complexes at lower γCD concentration region, and subsequently, these initially formed γCD bound AOH + dimers undergo disintegration to form a monomeric AOH + -γCD inclusion complexes at higher γCD concentration region. On the contrary, surprisingly, upon interaction with γCD, the neutral AO dye preferentially undergoes only dimeric AO-γCD complex formation throughout the entire host concentration used. The distinctly different supramolecular recognition of the AOH + and AO with γCD suggests that the difference in the protonation state of the dye and involvement of mainly hydrophobic driving forces in the complexation process plays the directing role in determining the overall distinctly different complexation behavior of the present AOH + -γCD and AO-γCD systems. Notably, the present study is the first report to reveal the pH dependent contrasting interaction mechanism of the different protonation states of the dye with the γCD host. The observed results may provide a suitable approach to design pH-controlled supramolecular assemblies, potentially applying to drug delivery, molecular sensor, molecular devices, logic gates, and stimuli-responsive functional material. Present study demonstrates the remarkable pH-dependent contrasting interaction mechanism of the protonated (AOH + ) and neutral (AO) forms of the acridine orange dye upon complexation with γ-cyclodextrin (γCD) host.