Ca2+ and Mg2+ are inevitably introduced into the production backwater when the mineral processing plants treat wastewater with precipitation agent, both of them would affect the flotation effect of molybdenite. The different influence of Ca2+ and Mg2+ on the flotation of the molybdenite was compared and investigated by flotation experiments of pure minerals, measurements of zeta-potential, X-ray photoelectron spectroscopy (XPS) and solution chemistry of Ca2+ and Mg2+. The results showed that both Ca2+ and Mg2+ could be adsorbed on the surface of the molybdenite, which changed the surface zeta-potential of the molybdenite and inhibited the floatability of the molybdenite. The inhibition was stronger with the increase of the pH value. Under the alkaline condition, the inhibition of Mg2+ on floatability of the molybdenite was stronger than that of Ca2+. And when the pH value was more than 9.0, the inhibition difference between the two ions was observed. When pH=11.0, the recovery of Mg2+ with a concentration of 800 mg/L decreased by about 34 percentage points compared with that of Ca2+ with a concentration of 800 mg/L. Both Ca2+ and Mg2+ could combine with MoO42– on the “edge” of molybdenite and form precipitation of molybdate salt, which existed on the surface of molybdenite in the form of chemical adsorption. When pH value was more than 9.15, calcium still existed in the form of Ca2+ and Ca(OH)+ and adsorbed on the surface of molybdenite, and the zeta-potential of the molybdenite in slurry containing Ca2+ showed an increasing tendency with the increase of the pH value. While magnesium deposit on the surface of molybdenite in the form of Mg(OH)2, and the zeta-potential of the molybdenite in pulp containing Mg2+ decreased rapidly with the increase of the pH value. This is the main reason for the difference between Ca2+ and Mg2+ in molybdenite?s floatability inhibition.