The secondary, ternary and quaternary structures of the biological ion channel Gramicidin-A are investigated by means of UV-visible and fluorescence spectroscopies, and by computational simulations in water/methanol solvent with various molar ratios. Both experiments and molecular dynamics calculations show that the two considered conformations, i.e. the head-to-head and double-stranded β-helix dimers, behave differently with regard to their stability as a function of the methanol content in the water/methanol mixture. To sum-up, calculations show that the double-stranded dimer is stabilized by methanol, even at low content, while the head-to-head dimer conformation breaks into two monomers which remain however coupled whatever the water/methanol mixture. In pure water and low methanol content, the resulting single β-helix structure is maintained due to the stabilization of the channel by water molecules and cations.