Efficient and selective hydrolysis of inert peptide bonds is of paramount importance. MOF-808, a metal–organic framework based on Zr₆ nodes, can hydrolyze peptide bonds efficiently under biologically relevant conditions. However, the details of the catalyst structure and of the underlying catalytic reaction mechanism are challenging to establish. By means of DFT calculations we first investigate the speciation of the Zr₆ nodes and identify the nature of ligands that bind to the Zr₆O₈H_4−x core in aqueous conditions. The core is predicted to strongly prefer a Zr₆O₈H₄ protonation state and to be predominantly decorated by bridging formate ligands, giving Zr₆(μ₃-O)₄(μ₃-OH)₄(BTC)₂(HCOO)₆ and Zr₆(μ₃-O)₄(μ₃-OH)₄(BTC)₂(HCOO)₅(OH)(H₂O) as the most favorable structures at physiological pH. The GlyGly peptide can bind MOF in several different ways, with the preferred structure involving coordination through the terminal carboxylate analogously to the binding mode of formate ligand. The pre-reactive binding mode in which the amide carbonyl oxygen coordinates the metal core lies 7 kcal higher in free energy. The preferred reaction pathway is predicted to have two close-lying transition states, either of which could be the rate-determining step: nucleophilic attack on the amide carbon atom and C–N bond breaking, with calculated relative free energies of 31 and 32 kcal mol⁻¹, respectively. Replacement of formate by water and hydroxide at the Zr₆ node is predicted to be possible, but does not appear to play a role in the hydrolysis mechanism.