pH regulators play a key role in mineral flotation. In the study, the effects of CaO, Na2CO3 and NaOH on the flotation separation of sphalerite and dolomite were investigated, and the interaction mechanisms were analyzed by flotation tests of mixed minerals, scanning electron microscope (SEM) and energy dispersion spectrum (EDS), turbidity, Zeta potential and the extended Derjaguin-Landau-Verwey-Overbeek (EDLVO) theory. The results indicated that Na2CO3 was favorable for flotation separation of sphalerite and dolomite, but NaOH had a negligible effect. CaO improved the flotation recovery of MgO to a maximum value of 9.29%, but it was unfavorable to the enriched sphalerite. SEM-EDS results revealed that dolomite was adsorbed on the sphalerite surface in the three systems, but the amounts of dolomite attached in the CaO system was higher than those of the other two systems. Turbidity tests indicated that the turbidity in the CaO system was less than that in Na2CO3 and NaOH systems. The sphalerite surface was negatively charged regardless of the pH regulators. Na2CO3 and NaOH hardly affected the surface potentials of dolomite. However, CaO changed the surface potential of dolomite from negative value to positive value under high alkaline condition. EDLVO theoretical calculations revealed that the electrostatic forces in CaO system were stronger than that in Na2CO3 and NaOH systems, leading to the slime coating.