Copper and lead are two important and widely used metals in industry. Chalcopyrite (CuFeS 2 ) is associated with galena (PbS) in ore, and it has been a research hotspot in separating galena from chalcopyrite by flotation. In this study, the flotation behaviors of chalcopyrite and galena were studied through flotation tests, adsorption measurements, solution chemistry calculation, Fourier transform infrared spectroscopy (FTIR) and molecular dynamics (MD) simulations. The results show that the floatability of chalcopyrite is better than that of galena in the presence of O-isopropyl-N-ethyl thionocarbamate (IPETC), and the recovery difference between chalcopyrite and galena is about 20% when IPETC is 7 × 10 −4 mol/L at pH 9.5, while the floatability difference between the two minerals is significant. Competitive adsorption of OH − and IPETC on mineral surfaces leads to lower floatability of galena than that of chalcopyrite. IPETC is able to remove the hydration layer on mineral surfaces and then adsorb on active sites. The floatability of minerals is enhanced with the increase of their hydrophobicity. This study provides a reference to separate galena from chalcopyrite.