ABSTRACT In seawater flotation, some chemical properties of seawater affect the flotation separation of chalcopyrite and sphalerite. Using seawater instead of fresh water to separate copper – zinc can save fresh water resources and reduce lime consumption, which has broad prospects in terms of economic and environmental protection. In this paper, the flotation separation behavior of chalcopyrite and sphalerite in seawater was tested and the mechanism was analyzed. As a contrast, the copper grade of concentrate in seawater flotation declined from 23.7% to 22.6%. Meanwhile, the recovery of chalcopyrite declined from 66.2% to 42.8% and the SI value declined from 2.210 to 1.599. With the addition of sodium hexametaphosphate, sodium silicate and EDTA, the SI value increased from 1.599 to 2.176, 2.238 and 2.096, respectively. Nevertheless, when EDTA was excessive, the SI value was only 1.998. This indicated that excessive EDTA is unfavorable to flotation separation of chalcopyrite from sphalerite. Wettability analysis, zeta potential analysis, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, density functional theory calculation and E-DLVO calculation verified the flotation separation behavior and revealed the mechanism.