To scrutinise the impact of electric fields on the structure and vibrations of biomolecules in the presence of water, we study the sequential solvation of lithium diglycine up to three water molecules with cryogenic infrared action spectroscopy. Conformer-specific IR–IR spectroscopy and H₂O/D₂O isotopic substitution experiments provide most of the information required to decipher the structure of the observed conformers. Additional confirmation is provided by scaled harmonic vibrational frequency calculations using MP2 and DFT. The first water molecule is shown to bind to the Li⁺ ion, which weakens the electric field experienced by the peptide and as a consequence, also the strength of an internal NH⋯NH₂ hydrogen bond in the diglycine backbone. The strength of this hydrogen bond decreases approximately linearly with the number of water molecules as a result of the decreasing electric field strength and coincides with an increase in the number of conformers observed in our spectra. The addition of two water molecules is already sufficient to change the preferred conformation of the peptide backbone, allowing for Li⁺ coordination to the lone pair of the terminal amine group.