AbstractGlucosamine 6‐phosphate synthase converts fructose‐6P into glucosamine‐6P or glucose‐6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40‐kDa C‐terminal domain, which has already been characterized crystallographically. Now the three‐dimensional structures of the complexes with the reaction product glucose‐6P and with the transition state analog 2‐amino‐2‐deoxyglucitol‐6P have been determined. Glucose‐6P binds in a cyclic form whereas 2‐amino‐2‐deoxyglucitol‐6P is in an extended conformation. The information on ligand‐protein interactions observed in the crystal structures together with the isotope exchange and site‐directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine‐6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the ‐amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate.