Tragacanth gum (TG) was explored as a depressant to realize the flotation separation of molybdenite and talc. The flotation experiments indicated that when using potassium butyl xanthate (PBX) as a collector, molybdenite showed excellent floatability while talc was completely depressed by TG, thus realizing the flotation separation of the two minerals. X-ray photoelectron spectroscopy (XPS) analysis results showed that TG was adsorbed on molybdenite surface via chemisorption. The results of contact angle measurement, Fourier transform infrared (FTIR) spectroscopy, and time-of-flight secondary ion mass spectrometry (ToF-SIMS) indicated that the pre-adsorption of TG on molybdenite could not hinder the further chemisorption of PBX on molybdenite. Because PBX has no collecting ability on talc, the flotation separation of molybdenite and talc came true using PBX to collect molybdenite and TG to depress talc.