The dissolution characteristics of minerals, dissolution of flotation agents in solutions, and equilibrium of dissociations and associations serve as the basis for determining the optimal conditions for the effective components of flotation agents and for evaluating the interaction between flotation agents and minerals. This basis provided the theoretical support for the flotation separation of minerals. Based on this, the flotation separation of magnesite and calcite was realized using sodium dihydrogen phosphate, also known as monosodium phosphate (MSP), as a regulator and dodecylamine (DDA) as a collector. When MSP was used in the DDA system, single-mineral and binary mixed-ore flotation tests revealed that the floatability of calcite was significantly greater than that of magnesite and the separation of magnesite and calcite was more effective, respectively. Zeta potential measurements showed that MSP-containing negative groups could selectively reduce the zeta potential of calcite and promote the adsorption of DDA-containing positive groups on the surface of the calcite. However, this effect was negligible on the zeta potential of magnesite. Due to the stronger affinity of MSP to Ca2+ than that to Mg2+, as demonstrated by Fourier transform infrared and X-ray photoelectron spectroscopy analyses, the MSP was adsorbed onto the surface of calcite primarily by hydrogen bonds rather than magnesite, which promoted the stronger adsorption of DDA-containing positive groups on the surface of the calcite. As a result, the differences in the floatability of magnesite and calcite were enlarged by MSP. Thus, MSP can be utilized an effective regulator for the efficient separation of magnesite from calcite via reverse flotation.