Abstract: The environment-friendly and efficient selective separation of chalcopyrite and molybdenite poses a challenge in mineral processing. In this study, gum arabic (GA) was initially proposed as a novel depressant for the selective separation of molybdenite from chalcopyrite during flotation. Micro-flotation results indicated that the inhibitory capacity of GA was stronger toward molybdenite than chalcopyrite. At pH 8.0 with 20 mg/L GA addition, the recovery rate of chalcopyrite in the concentrate obtained from mixed mineral flotation was 67.49% higher than that of molybdenite. Furthermore, the mechanism of GA was systematically investigated by various surface characterization techniques. Contact angle tests indicated that after GA treatment, the hydrophobicity of the molybdenite surface significantly decreased, but that of the chalcopyrite surface showed no apparent change. Fourier transform-infrared spectroscopy and X-ray photoelectron spectroscopy revealed a weak interaction force between GA and chalcopyrite. By contrast, GA was primarily adsorbed onto the molybdenite surface through chemical chelation, with possible contributions from hydrogen bonding and hydrophobic interactions. Pre-adsorbed GA could prevent butyl xanthate from being adsorbed onto molybdenite. Scanning electron microscopy–energy-dispersive spectrometry further indicated that GA was primarily adsorbed onto the “face” of molybdenite rather than the “edge.” Therefore, GA could be a promising molybdenite depressant for the flotation separation of Cu–Mo.