Cholinesterases are well‐known and widely studied enzymes crucial to human health and involved in neurology, Alzheimer's, and lipid metabolism. The protonation pattern of active sites of cholinesterases influences all the chemical processes within, including reaction, covalent inhibition by nerve agents, and reactivation. Despite its significance, our comprehension of the fine structure of cholinesterases remains limited. In this study, we employed enhanced‐sampling quantum‐mechanical/molecular‐mechanical calculations to show that cholinesterases predominantly operate as dynamic mixtures of two protonation states. The proton transfer between two non‐catalytic glutamate residues follows the Grotthuss mechanism facilitated by a mediator water molecule. We show that this uncovered complexity of active sites presents a challenge for classical molecular dynamics simulations and calls for special treatment. The calculated proton transfer barrier of 1.65 kcal/mol initiates a discussion on the potential existence of two coupled low‐barrier hydrogen bonds in the inhibited form of butyrylcholinesterase. These findings expand our understanding of structural features expressed by highly evolved enzymes and guide future advances in cholinesterase‐related protein and drug design studies.