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The flotation separation of chalcopyrite from pyrite has attracted increasing attention due to the consumption of vast water resources and depressants. This study proposed the seawater oxidation pretreatment for non-depressant flotation separation of chalcopyrite from pyrite, as an effective and environmentally friendly strategy. Without the addition of depressants, seawater oxidation for 3 d effectively depressed pyrite flotation, with the highest recovery difference greater than 70% and a selectivity index greater than 6 between chalcopyrite and pyrite. The surface investigation showed that pyrite surface was more readily oxidized to form hydrophilic Fe oxidants/oxyhydroxides, as compared to that of chalcopyrite. Further UV-visible spectrophotometer and Fourier transform infrared spectrum (FTIR) results indicated that xanthate was less adsorbed onto the treated pyrite surface, resulting in un-floatable particles. Chalcopyrite surface was changed slightly due to seawater oxidation, thereby insignificantly affecting its flotation. The coordination theory was further used to reveal the combination mechanisms between xanthate and pyrite or chalcopyrite. This study therefore provides a promising strategy to effectively separate chalcopyrite from pyrite, especially in the freshwater-deficient area.