Pyrite, a gangue mineral, is commonly associated with valuable base metal sulfide minerals and the separation of pyrite from these valuable sulfide minerals by flotation is difficult. In this study, a type of biopolymers, modified from lignosulfonates, was used to reject pyrite in chalcopyrite flotation based on the previous investigations. It was found that these biopolymers selectively depressed pyrite flotation with little effect on chalcopyrite flotation. The presence of pyrite decreased the depression effect of biopolymers on chalcopyrite flotation due to the preferential adsorption of biopolymers on copper-activated pyrite as revealed by the adsorption isotherms and ToF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry) analyses. The presence of chalcopyrite increased the depression effect of biopolymers on the flotation of copper-activated pyrite due to enhanced oxidation of the copper activation product, Cu(I)S, on pyrite as a result of the galvanic interaction among Cu(I)S, pyrite and chalcopyrite.