Cu2+ and Fe3+ are generally inevitable in the flotation of molybdenite. In this work, the effect of Cu-2 and Fe3+ on the flotation of molybdenite was investigated through flotation tests. The influence mechanism was studied by Zeta potential measurement, scanning electron microscopy-energy-dispersive spectroscopy (SEM-EDS), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analysis. The results showed that the flotation of molybdenite was strongly depressed by Cu2+ and Fe3+, and the depression of Fe3+ was more powerful than that of Cu2+. The depression performance of molybdenite was significantly influenced by pH in the presence of Cu2+ and Fe3+, which was due to the adsorption of different hydrolyzed species as pH varied. The depression mechanism involved two different models in Cu2+ solution. In acid media, the depression was mainly contributed to the coverage of less hydrophobic CuS, as well as a small amount of hydrophilic Cu(OH)(2) and Cu2+ on molybdenite surface. While in alkaline condition, the adsorption of Cu(OH)(2) precipitate was the main reason for the depression. In Fe3+ solution, Fe(OH)(3) precipitate was the main component adsorbed on molybdenite surface to make the surface of molybdenite hydrophilic.