Abstract: This study investigated the effect of konjac glucomannan (KGM) on the flotation separation of calcite and scheelite. Micro-flotation tests showed that under the action of 50 mg/L KGM, the floatability of calcite notably decreased, while the impact on scheelite was negligible, resulting in a recovery difference of 82.53%. Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) analyses indicated the selective adsorption of KGM on the calcite surface. Test results of the zeta potential and UV-visible absorption spectroscopy revealed that KGM prevented the adsorption of sodium oleate on the calcite surface. X-ray photoelectron spectroscopy (XPS) analysis further confirmed the chemical adsorption of KGM on the calcite surface and the formation of Ca(OH)₂. The density functional theory (DFT) simulation results were consistent with the flotation tests, demonstrating the strong adsorption performance of KGM on the calcite surface. This study offers a pathway for highly sustainable and cost-effective mineral processing by utilizing the unique properties of biopolymers such as KGM to separate valuable minerals from gangue minerals.