When a solvent molecule is replaced by another molecule with larger proton affinity, the strength of all other hydrogen bonds decreases. This is the concept of anticooperativity by successive substitution in a mixed solvation system. In the present study, this concept is demonstrated in H+(CH3OH)(m)(H2O)(n) (m + n = 5 and 6) mixed clusters by a joint theoretical and infrared (IR) spectroscopic approach. The observed IR spectra of the mixed clusters exhibit two high-frequency shifts of hydrogen-bonded OH stretch bands with increasing methanol content. These trends are well reproduced by first-principle IR spectra simulated by thermal averaging over a set of configurational isomers under the quantum harmonic superposition approach. Theoretical analysis on the magnitude of charge transfer from the protonated site to the solvent molecules is found to be in agreement with the spectroscopic measurement that the individual hydrogen bond in the mixed clusters is weakened with an increase of the mixing ratio of methanol to water.