With the advancement of green mine construction and the increasing difficulty of lead–sulphur resources, the traditional lime high-alkalinity lead–sulphur separation technology has exposed many shortcomings. In this study, based on the galvanic interaction of galena and pyrite, an environmental-friendly depressant dithiothreitol (DTT) was used to separate pyrite and galena under low alkalinity. In addition, the mechanism of DTT was revealed by electrochemical, ion dissolution, contact angle, zeta potential and DFT calculation. The results indicated that the galvanic interaction between galena and pyrite promoted the oxidation of galena and the dissolution of Pb2+ from the surface of galena, and Pb2+ would be adsorbed on the pyrite surface and increase the floatability of pyrite. DTT could selectively depress pyrite at low alkalinity. The interaction mechanism of DTT was that DTT could weaken the galvanic interaction of galena and pyrite and complex lead ions. In addition, DTT was adsorbed on the pyrite surface through the Fe-S bond, and the pre-adsorbed DTT hindered the adsorption of DDTC on the pyrite surface, thus the pyrite flotation was depressed.