Humic acid (HA) has been studied as a potential selective depressant for molybdenite in the flotation separation of molybdenite and talc in this paper. In the single mineral flotation tests, HA has shown good depression ability to molybdenite over the entire pH range tested from pH 3-11, while the floatability of talc was only affected by HA under strong acidic condition (pH 3). In the mixed minerals flotation, HA can effectively depress the flotation of molybdenite while not affecting the flotation of talc under strong alkaline condition (pH 11). Contact angle measurements have proved that the hydrophobicity of both talc and molybdenite decreases after treatment by HA, however, the contact angles of treated talc are still higher than molybdenite. Equilibrium adsorption studies indicate that HA has a much higher adsorption density on molybdenite than on talc. The preferential adsorption of HA on molybdenite was further confirmed by diffusion reflectance infrared Fourier transform spectroscopy (DRIFTS) and atomic force microscopy (AFM) imaging. All of the experimental results suggest that humic acids can be employed as a selective depressant for molybdenite during the flotation separation of molybdenite and talc under alkaline conditions, and the higher adsorption density of HA on molybdenite over talc is the primary reason for the selective separation.