We previously reported that gigantol extracted from Caulis Dendrobii has significant therapeutic benefits for the treatment of galactosemic cataracts through its ability to inhibit aldose reductase (AR) activity. In this study, we identified the binding sites and structurally characterized the interaction between gigantol and AR, to understand the mechanism (s) of the effects of gigantol on cataracts. Gigantol was found to be protective against diabetic cataracts (DC) in rats induced by streptozotocin. Molecular docking predicted the binding sites between AR and gigantol to be residues Trp111, His110, Tyr48 and Trp20. Mutation of each of these residues led to a significant reduction in AR activity. Cold-spray ionization mass spectrometry measurements showed that the binding of gigantol to AR is oncentration-dependent and that the maximum stoichiometric ratio of non-covalent bonding is 1:24.4. pH and temperature did not influence the interaction. Taken together, we provide further mechanistic evidence of the beneficial effects of gigantol on DC.