Silicate minerals with a certain degree of flotability are often easy to mix into sulfide ore concentrate in mineral processing industry. In this paper, the adsorption of yeast dextran on clinochlore and its application in pyrite/clinochlore separation were investigated. The adsorbed amount and micro polarity measurement results displayed that the yeast dextran molecules selectively adsorbed onto clinochlore surface compared with pyrite. The adsorbed yeast dextran resulted in the increase in the surface polarity of clinochlore surface, and inhibited the further adsorption of xanthate, thus keep it hydrophilic and depressed. Quantum chemical computation results indicated that yeast dextran was mainly adsorbed on mineral surface by the chelation with the surface metal active sites, and the chelating strength of yeast dextran with three ions was in the sequence of Fe³⁺ > Mg²⁺ > Fe²⁺. While Mg²⁺, Fe²⁺ and Fe³⁺ are the main metal ions on the surface of clinochlore, and Fe²⁺ is the unique metal ions on pyrite surface. This is the reason of the selectivity of the yeast dextran depressant for pyrite/clinochlore flotation system. The flotation results demonstrated that yeast dextran was qualified to selectively depress clinochlore in pyrite flotation.