The imidazole 15N signals of histidine 64 (His‐64), associated with the proton‐transfer function of human carbonic anhydrase II (hCAII), were observed [1]. Using the pH‐dependency of ring 15N signals and a comparison between experimental and simulated curves, we determined that the tautomeric equilibrium constant (KT) of His‐64 is 1.0. This unique value characterizes the nitrogen atoms of the His‐64 ring as both a general acid and base. Based on this feature, we suggest that the tautomerization of His‐64 can mediate the transfer of protons at a neutral pH with high efficiency. Comparing the KT of His‐64 with that of the histidine analog compounds shows that the imidazole group intrinsically tends to be the Nε2‐H tautomer in aqueous solution, unless hydrogen bond interacts with δ1‐nitrogen. This clarifies the structure ‐specifically, the hydrogen bonding pattern of His‐64. As a result, we propose that the formation and decay of hydrogen bonds should occur in the water bridge between His‐64 and zinc‐bound water during hydration and dehydration reactions in catalysis. This provides a new insight into the proton‐transfer mechanism in hCAII, i.e., a reorganization or flow of water molecules occurs in the water bridge when His‐64 is the Nε2‐H tautomer. During this conference, extensively low field shifts of 1H signals and theoretical consistency will be also discussed.