There is a successful use of protamine-insulin formulation (Zn+insulin+protamine) to treat diabetes in which protamine is added to the stable form of hexameric insulin (Zn-insulin). The biophysical characterization of Zn-insulin, which can dissociate to form biologically active monomers, is well reported. However, its interaction with protamine, which is at the core of the mode of action in many pharmaceutical formulations, is unresolved. Through biophysical characterization, we have tried to dissect the interactions driving the Zn-insulin-protamine complexation. Based on the thermal melting study, it was found that protamine indulges in the destabilization of Zn-insulin. Fluorescence results revealed that Zn-insulin experiences Tyr quenching in the presence of protamine, undergoing a significant conformational change. As shown by the molecular docking study, protamine disturbs the H-bonding network at the dimer interface by binding to the amino acid residues involved in the dimer stabilization. It may result in the freeying of B-chain, introducing conformational fluctuations in the insulin. This is well supported by the loss of helical content seen in circular dichroism. Further, the insulin-protamine complex formation was strongly dominated by hydrogen bonding and a few hydrophobic contacts. The endothermic heat and positive entropy observed in isothermal titration calorimetry in the dissociation of Zn-insulin-protamine is a reflection of that. Finally, the ANS binding study proposed the adaption of a flexible conformation by the Zn-insulin-protamine complex containing exposed hydrophobic residues, a potential arrangement for successful receptor binding.