Since the excessive dosage of Pb2+ ions could facilitate the gangue mineral quartz activation and cause serious environmental problems, this work has an attempt to examine the performance of cupferron as a new alternative collector in selective separation of cassiterite from quartz with low dose of Pb2+ ions. Microflotation results show the improved selectivity of cupferron towards cassiterite as compared to quartz. Accordingly, its selective adsorption mechanism has been investigated by zeta potential measurements, X-ray photoelectron spectroscopy (XPS) analysis and first-principle density functional theory (DFT) calculations. The results of zeta potential measurements demonstrate the stronger adsorption of cupferron on cassiterite surface than quartz whether Pb2+ ion was added or not. The XPS analysis and first-principle DFT calculations consistently show that Sn4+ ions on the hydrated cassiterite surface exhibit much stronger reactivity towards cupferron adsorption than Si atoms on the hydrated quartz surface, resulting in a remarkable difference between the collecting ability of cupferron towards cassiterite and quartz. This work should shed new light on the discovery of cleaner flotation reagents for cassiterite beneficiation.