The D-aldohexose dehydrogenase from the thermoacidophilic archaeon Thermoplasma acidophilum (AldT) is a homotetrameric enzyme that catalyzes the oxidation of several D-aldohexoses, especially D-mannose. AldT comprises a unique C-terminal tail motif (residues 247-255) that shuts the active-site pocket of the neighboring subunit. The functional role of the C-terminal tail of AldT has been investigated using mutational and crystallographic analyses. A total of four C-terminal deletion mutants (Delta254, Delta253, Delta252, and Delta249) and two site-specific mutants (Y86G and P254G) were expressed by Escherichia coli and purified. Enzymatic characterization of these mutants revealed that the C-terminal tail is a requisite and that the interaction between Tyr86 and Pro254 is critical for enzyme activity. The crystal structure of the Delta249 mutant was also determined. The structure showed that the active-site loops undergo a significant conformational change, which leads to the structural deformation of the substrate-binding pocket.