Herein, we have explored the conformational alterations of hemoglobin (Hb) in presence of a cleavable gemini surfactant (C16-C402-C16). The concerned surfactant was found to induce significant structural perturbations in Hb. UV-vis spectroscopy, steady-state/time-resolved fluorescence, and other utilized techniques have authenticated the complexation of Hb with the gemini surfactant. CD has demonstrated the alterations in secondary structural elements (alpha-helicity, beta-sheet, beta-turn, and random coil) of Hb upon C16-C402-C16 addition. Atomic force microscopy (AFM) has revealed the existence of unique star-shaped gemini surfactant microstructures aligned to Hb in a necklace pattern. The H-1 NMR peak broadening and lower delta values hint at the binding of the concerned gemini surfactant to Hb. Molecular docking and DFT calculations have further substantiated the Hb-gemini complex formation and the involvement of electrostatic/hydrophobic forces therein. In future, these results might pave-the-way to construct self-assembled, sustainable, and green surfactant-protein mixtures for their end-use in industrial, engineering, biomedical, drug delivery, gene transfection, and other relevant excipient formulations. (C) 2018 Elsevier Inc. All rights reserved.