Abstractd‐glucopyranose undergoes mutarotation into a mixture of anomers, α‐ and β‐d‐glucopyranose, once it is dissolved in an aqueous solution. Distinguishing the two anomers by routine NMR methods are difficult because of their similarity and hence overlapped 1H‐NMR spectra. In this paper, we demonstrate that the DOSY technique can be used to discern the two anomers of the d‐glucopyranose. This is possible when taking advantage of a matrix effect of Ca2+, which separate the α‐ and β‐ anomers of d‐glucopyranose by their differences in diffusion. The effect of Ca2+ was further supported by DFT calculations. Our results demonstrate the power of DOSY NMR to gain a deeper understanding of the interactions between carbohydrates and metal ions.