The separation of molybdenite and chalcopyrite is a major challenge because of their similar floatability. It is essential to discover a depressant for selectively inhibiting chalcopyrite in a profitable and sustainable way. A novel chalcopyrite depressant, thiolactic acid (TLA), was proposed. The flotation behaviors were studied through micro-flotation and artificial mixed minerals flotation. Micro-flotation experiments indicated that thiolactic acid could significantly suppress chalcopyrite recovery from 81% to 9.7%, along with molybdenite recovery always more than 71%. Artificial mixed minerals flotation experiments showed a distinct separation behavior between chalcopyrite and molybdenite with an optimal Gaudin’s selectivity index of 12.9. The reactivity mechanism of TLA on molybdenite and chalcopyrite surfaces was determined by FT-IR, XPS, and AFM with a possible adsorption model presented. The results revealed that —SH and —COOH groups of TLA preferentially occupied the active copper site on chalcopyrite surface, inhibiting subsequent sodium n-butyl xanthate chemisorption.