A novel quaternary ammonium-based cationic gemini surfactant (S6) having 1,6 di-bromo hexane as a spacer, have been used and its interaction with insulin in aqueous solution (pH, 7.40) was investigated by several methods including fluorescence spectroscopy, UV-Vis spectroscopy, circular dichroism, dynamic light scattering, ζ-potential measurements, conductivity and transmission electron microscopy. Conductometry and fluorescence studies confirmed the complex formation between S6 and insulin. TEM micrographs revealed that the S6 micelles were of spherical shape with size distribution between 101-140 nm. The critical micelle concentration and some physicochemical properties were determined by conductance measurements. Fluorescence quenching studies in the presence of acrylamide indicated that in the protein-surfactant interaction the solvent accessibility of Tyr residues is reduced. Furthermore CD experiments (far- and near-UV CD) showed that, the content of alpha-helix increases with increasing S6 concentration and some conformational changes occur in protein structure. The results from dynamic light scattering and ζ-potential measurements showed that insulin charge neutralization and hexamer dissociation take place in the presence of surfactant. Altogether, the S6 cationic Gemini surfactant can be considered as a candidate for insulin delivery.