This work focuses on the organic depressant, disodium bis(carboxymethyl) trithiocarbonate (DBT), as a selective depressant in copper-molybdenum sulfide flotation separation. Micro-flotation, Zeta potential, FTIR and XPS measurements were carried out to investigate the selective depression mechanism of DBT on chalcopyrite. Zeta potential and FTIR measurements revealed that DBT had higher affinity for chalcopyrite than molybdenite and the XPS results of chalcopyrite before and after treatment with DBT further proved that DBT adsorbed on chalcopyrite surface. The investigation indicates that the mechanism of DBT adsorbing on chalcopyrite is mainly physical adsorption. Locked circuit experiments were carried out and the results showed that DBT could be considered as a cleaner option in commercial Cu-Mo flotation separation circuits.