The flotation separation of chalcopyrite from monoclinic pyrrhotite using food-grade guar gum (FGG) as a depressant was studied through flotation tests, kinetic studies, dynamic potential measurements, adsorption experiments, and infrared spectral analyses. The microflotation results showed that the flotation separation of chalcopyrite from monoclinic pyrrhotite could not be realized by adding mixed aerofloat (CSU11) alone. The depressant FGG exhibited a selective depression effect on monoclinic pyrrhotite by controlling the pulp pH range from 5.0 to 6.0, with a maximum floatability variation of 79.36% in the presence of CSU11. The flotation kinetics, zeta-potential, adsorption, and infrared spectroscopy studies revealed that the FGG could absorb more strongly on the surface of monoclinic pyrrhotite than on the surface of chalcopyrite. In addition, the results revealed that the interaction of FGG with the monoclinic pyrrhotite surface was governed primarily by strong chemisorption, whereas FGG mainly bonded to chalcopyrite through hydrogen bonding. This difference was responsible for the excellent depression selectivity of FGG toward monoclinic pyrrhotite flotation and weak depression effect toward chalcopyrite flotation.