In this study, hydroxypropyltrimethyl ammonium chloride chitosan (HACC) was first introduced as a depressant during separating chalcopyrite from molybdenite (Cu-Mo). The selective effects of HACC on the separation of Cu-Mo were conducted by single-mineral flotation experiments. The findings from this study revealed that HACC helped separate Cu and Mo efficiently at pH 6 with 8 mg/dm3 of HACC, resulting in 76.22% and 5.38% of Mo and Cu flotation recovery, respectively. The adsorption mechanism of HACC was investigated via zeta potential, adsorption density, and contact angle measurement along with FT-IR and XPS analyses. The contact angle and adsorption density measurements offer indisputable proof that HACC can adsorb on the surface of chalcopyrite. Furthermore, FT-IR and XPS analyses confirm that N atoms in quaternary ammonium groups of HACC interact with Cu sites on the surface of chalcopyrite. The findings also suggest that HACC adsorbs on the surface without significantly impacting molybdenite. All these results confirm that HACC can be an effective chalcopyrite depressant.