Herein, we have investigated the effect of microhydration on the secondary structure of a capped dipeptide Boc-DPro-Gly-NHBn-OMe (Boc = tert-butyloxycarbonyl, Bn = Benzyl), i.e., Pro–Gly (PG) with a single H2O molecule using gas-phase laser spectroscopy combined with quantum chemistry calculations. Observation of a single conformer of the monohydrated peptide has been confirmed from IR-UV hole-burning spectroscopy. Both gas-phase experimental and theoretical IR spectroscopy results confirm that the H2O molecule is inserted selectively into the relatively weak C7 hydrogen bond (γ-turn) between the Pro C=O and NHBn N–H groups of the peptide, while the other C7 hydrogen bond (γ-turn) between the Gly N–H and Boc C=O groups remains unaffected. Hence, the single H2O molecule in the PG⋯(H2O)1 complex significantly distorts the peptide backbone without appreciable modification of the overall secondary structural motif (γ–γ) of the isolated PG monomer. The nature and strength of the intra- and inter-molecular hydrogen bonds present in the assigned conformer of the PG⋯(H2O)1 complex has also been examined by natural bond orbital and non-covalent interaction analyses. The present investigation on the monohydrated peptide demonstrates that several H2O molecules may be required for switching the secondary structure of PG from the double γ-turn to a β-turn that is favorable in the condensed phase.