In this paper, the depressant effect of the whey protein, a chelating agent for copper ions, on the flotation of copper-activated marmatite was investigated by micro-flotation, zeta potential, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FT-IR). The results showed that the formation of a complex through the interaction between the copper ions and the whey protein effectively prevented the activation of marmatite from copper ions, and the flotability of the copper activated marmatite was remarkably decreased. The finding can potentially offer new choices for developing new reagents for depressing the copper-activated marmatite in flotation separation of the polymetallic sulfide ores.