Vacuum ultraviolet (VUV) electronic circular dichroism (ECD) spectra of d-glucose, α-d-glucopyranose, and β-d-glucopyranose were measured in aqueous solution down to 163 nm using a synchrotron radiation VUV-ECD spectrophotometer and theoretically analyzed using molecular dynamics (MD) simulations with explicit water molecules and using time-dependent density functional theory (TDDFT). The theoretically calculated spectra reproduced the experimentally observed spectra well, revealing that VUV-ECD exhibited unique spectra depending on the α-anomer and β-anomer configurations of the hydroxyl group at C-1 and the three gauche (G) and trans (T) rotamer conformations (GT, GG, and TG) of the hydroxymethyl group at C-5. These unique spectra could be ascribed to differences in the patterns of intramolecular hydrogen bonds around the hydroxymethyl group at C-5 for the three rotamers and around the hydroxyl group at C-1 for the two anomers. The strengths of these intramolecular interactions increased as the degree of hydration around the corresponding chromophores decreased, suggesting that hydration is a key factor for stabilizing rotamer and anomer structures. The rotamerization and anomerization mechanisms are further discussed in terms of differences in the intramolecular interactions and the degree of hydration among the rotamer and anomer structures. The findings demonstrate that VUV-ECD spectroscopy is a useful tool for characterizing the equilibrium structures of monosaccharides.