Abstract: In the work, DL-2,3-diaminopropionic acid hydrochloride (DL-DAP) was used as a depressant of chalcopyrite for the flotation separation of chalcopyrite and molybdenite. Results of micro-flotation tests demonstrated that DL-DAP significantly depressed chalcopyrite flotation, but had negligible impact on that of molybdenite. Multiple analytical methods—including zeta potential measurements, contact angle analysis, solution chemistry calculations, FTIR spectroscopy, and X-ray photoelectron spectroscopy (XPS)—were employed to elucidate the depression mechanism. DL-DAP selectively depressed chalcopyrite by increasing its surface electronegativity and hydrophilicity. The amino groups of DL-DAP formed coordination complexes with metal sites (Cu/Fe) on the chalcopyrite surface. Within these complexes, the hydrophilic carboxyl groups of DL-DAP were oriented outward, exposing to the aqueous phase and thereby enhancing the hydrophilicity of chalcopyrite surface and causing its depression. These findings provided a theoretical basis for understanding the role of amino-based depressants in the flotation separation of sulfide minerals.