Abstract: The environment-friendly and efficient selective separation of chalcopyrite and molybdenite presents 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 in flotation processes. The micro-flotation results indicated that GA exhibited a stronger inhibitory capacity towards molybdenite rather than chalcopyrite. At pH 8.0 with the addition of 20 mg/L GA, the recovery of chalcopyrite in the concentrate obtained from mixed minerals flotation was 67.49% higher than that of molybdenite. Furthermore, the mechanism of GA was systematically investigated by various surface characterization techniques. The contact angle tests indicated that the hydrophobicity of molybdenite surface significantly decreased after GA treatment, while there was no apparent change in the hydrophobicity of chalcopyrite surface. The Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) results indicate that the interaction force between GA and chalcopyrite was weak. In contrast, GA primarily adsorbed onto the surface of molybdenite through chemical chelation, with hydrogen bonding and hydrophobic interactions also possibly contributing. Pre-adsorbed GA could prevent BX from adsorbing onto the molybdenite. Scanning electron microscopy energy dispersive spectroscopy (SEM-EDS) analysis further indicated that GA primarily adsorbed onto the "face" of molybdenite rather than the "edge". Therefore, GA could be a promising molybdenite depressant and provide flotation separation of Cu–Mo.