It is difficult to realize an effective separating of chalcopyrite and pyrite without using any depressants due to their similar physicochemical properties. Herein, a cyclic oligosaccharide carboxymethyl-β-cyclodextrin (CMCD) with carboxyl and hydroxyl groups was introduced for the first time as a novel environmentally friendly depressant for separating chalcopyrite from pyrite. The depression performance and adsorption mechanism of CMCD in the flotation separation of chalcopyrite and pyrite were investigated through micro-flotation experiments, molecular dynamics (MD) simulations, and a series of characterization tests. The flotation findings manifested that under weakly acidic conditions (pH=6.5±0.2), a high-quality copper concentrate with a copper recovery of 73.15 % and a grade of 28.06 % was obtained. CMCD exhibited superior inhibition of pyrite compared with other traditional inhibitors. The adsorption density of CMCD onto the pyrite surface was significantly higher than that of chalcopyrite, and the pre-treatment with CMCD hindered the attachment of sodium butyl xanthate (SBX) on pyrite but had a minor effect on chalcopyrite, as confirmed by contact angle, adsorption amount, and Atomic Force Microscope analyses. Moreover, X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy analyses robustly demonstrated that the carboxyl and hydroxyl groups in the CMCD molecule primarily reacted with the metal hydroxides on the pyrite surface through hydrogen bonding, leading to a more hydrophilic surface. MD simulations further confirmed at the molecular level that the affinity of CMCD for the pyrite surface was much greater than for chalcopyrite. In summary, CMCD shows promising potential as a depressant for separating chalcopyrite from pyrite.