In this work, L-cysteine was introduced as an environmentally-efficient flotation reagent in the selective separation of molybdenite from chalcopyrite. Its flotation performance and adsorption mechanism onto chalcopyrite were also investigated by flotation experiment, zeta potential, FTIR spectra, Raman spectra, TOF-SIMS measurements and DFT calculations. The flotation results indicated that L-cysteine exhibited stronger depressing ability than the conventional chalcopyrite depressants of Na₂S, NaHS and sodium thioglycolate, suggesting it could become a promising potential depressant in the selective flotation separation of molybdenite from chalcopyrite. The results of zeta potential, FTIR spectra, TOF-SIMS and Raman spectra measurements demonstrated that L-cysteine might chemisorb on the Cu atoms of chalcopyrite surface via -SH and –NH₂ groups to form a stable L-cysteine-Cu chelate compound. The DFT calculations further supported the experimental characterizations; the calculated adsorption reaction energy of L-cysteine with Cu in the (0 0 1) surface of chalcopyrite was much lower than that of L-cysteine with Fe, which implied that L-cysteine would prefer to adsorb onto the Cu site of chalcopyrite surface.