AbstractNeutron diffraction analysis studies reported an isolated hydronium ion (H3O+) in the interior of d‐xylose isomerase (XI) and phycocyanobilin‐ferredoxin oxidoreductase (PcyA). H3O+ forms hydrogen bonds (H‐bonds) with two histidine side‐chains and a backbone carbonyl group in PcyA, whereas H3O+ forms H‐bonds with three acidic residues in XI. Using a quantum mechanical/molecular mechanical (QM/MM) approach, we analyzed stabilization of H3O+ by the protein environment. QM/MM calculations indicated that H3O+ was unstable in the PcyA crystal structure, releasing a proton to an H‐bond partner His88, producing H2O and protonated His88. On the other hand, H3O+ was stable in the XI crystal structure. H‐bond partners of isolated H3O+ would be practically limited to acidic residues such as aspartic and glutamic acids in the protein environment.