Solvent-protein interactions are important for protein biological functions, especially for a coupled folding and binding system such as insulin. By monitoring the change in the conformation of insulin dimers during dissociation with temperature-jump infrared spectroscopy, we show that co-solvents can significantly destabilize the dimers by perturbing their hydrophobic center. The transition from the native to intermediate dimer state is observed as the buried residues are exposed to solvents in the presence of 10% dimethyl sulfoxide and with α-helices unfolding when ethanol is present, which reduces the dissociation time dramatically to 50% and 20% of the value in a D2O solution, respectively. We propose a self-consistent analysis using complementary methods to resolve this coupled folding and binding process and obtain a much higher rate of monomer association than of intermediate folding. Our results demonstrate that the conformational changes are critical in dimer formation and strongly affected by co-solvents.