Ion-specific effects on peptides and proteins are crucial to the biomolecular structure. Compared with the significant role of anions, the subtle effects of cations on proteins have not been clearly understood. Importantly, divalent cations like Ca2+ and Mg2+ are known to be crucial to biological functions. Herein, we report that the direct binding of the divalent cations to the amide oxygen in aqueous solution triggers an amide???iminolate tautomer equilibrium. For N-methyl acetamaide (NMA) dissolved in aqueous 5M CaCl2 solution, the formation of an amide tautomer is strongly supported by two-dimensional infrared (2D IR) spectroscopy of the amide vibration and molecular dynamics (MD) simulations of molecular phenomenon. The interconversion timescale (~17 ps) between the tautomers confirms that Ca2+ forms direct contact with the amide O intermittently. These results confirm the arrest of an unusual amide tautomer by the divalent cations and provide an explanation for the shift in the aggregation pathway of neurotoxic peptides in the presence of divalent cations