Abstract We investigated the biosynthesis of the unique structure of calditol, a part of the characteristic lipid molecules in the major part of thermophilic archaea, Sulfolobus sp. Deuterium-labeling experiments with deuterated glucose substrates, were carried out. The results clarified that calditol was biosynthesized from glucose with C–C bond formation between C-1 and C-5, and no loss of deuterium at C-6 of glucose was observed. Two reaction mechanisms were possible when these experimental findings, and conventional reports were considered. Among these, the course that assumes C-4 oxidation as the starting point would be preferable from the resemblance to myo-inositol biosynthesis. Furthermore, the high deuterium incorporation at C-1 of calditol from C-1 of glucose suggests that activation, such as oxidation at C-1, is not involved in the ether C–O bonding.