The properties of low molecular weight glycolipids at subzero temperatures have been paid much attention due to their great potential for applications in cryopreservation. In this study, the ability of glycolipids to maintain their lyotropic properties was investigated by analyzing the aggregation behavior of octyl-β-Dglucoside (β-OGlu) during solidification and melting of electrolyte/ice eutectic mixtures using a simultaneous X-ray diffraction-differential scanning calorimetry measurement and cryomicroscopic observation. During the freezing process, eutectic formation increased the concentration of the β-OGlu micelle solution in the unfrozen phase, leading to ordering of the molecular assembly. The molecular assembly exhibited a reversible phase transformation between liquid crystalline phases (e.g., lamellar (Lα) and hexagonal (H) phases) and an isotropic micelle solution (I), depending on the degree of eutectic formation, which was regulated by temperature. Because the coexistence of electrolyte/ice eutectic often induces the dehydration of lipids, the persistence of lyotropic properties under such arid conditions demonstrates an attractive performance of glycolipids for use in low temperature applications, such as the cryopreservation of poorly water-soluble drugs or proteins.