Three types of chelating depressants were studied for chalcopyrite/pyrite separation, including S-S, S-O, and O-O types, via density functional theory calculations and microflotation. The calculation results indicate that the depressant’s chelating atoms have large coefficient and great activity according to the molecular frontier orbital (HOMO and LUMO) and the orbital coefficients. For S-S type of depressant, S atom in both keto or enol forms won’t affect their HOMO and LUMO patterns and the orbital contributions. For S-O type, the presence of N atom in the ring structure of a molecular will increase the reactivity of O-Cu while weak S-Cu. For O-O type, the electron supply capacity of benzene ring is higher than strain chain, and atom N in strain chain increased their electron supply capacity. The microflotation results basically confirmed the prediction based on the calculation. The simulation results demonstrate that the interaction of a depressant with metals and minerals are affected obviously by the spatial structure and electronic structure of an atom in its molecular.