BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the β-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the β-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (K D = 4.0 μM), followed by the formation of a covalent bond (rate constant of 0.163 s -1 ), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.