Abstract: To achieve efficient flotation separation of complex Cu-Zn-Fe sulfide ores, highly selective collectors play a crucial role. A novel azathione collector 2-hydroxybenzothiazole (HBT) was developed by substituting the exocyclic S atom of 2-mercaptobenzothiazole (MBT) with a highly electronegative O atom. Compared to the collectors MBT and SEX, under the conditions of pH = 8 and a dosage of 1 × 10⁻⁴ mol/L, HBT achieved superior selective flotation separation of chalcopyrite from sphalerite and pyrite. XPS analysis revealed that new peaks appeared in the N 1s and S 2p spectra on the chalcopyrite surface after treatment with HBT and MBT. DFT calculations demonstrated that both HBT and MBT can displace the weakly adsorbed water molecules on the chalcopyrite surface and form strong covalent bond adsorption, whereas they struggle to displace the strongly adsorbed water molecules on the surfaces of sphalerite and pyrite and only generate ionic bond adsorption. The higher electronegativity of the O atom in HBT makes it more difficult to donate electrons, and the adjacent imino group carries a lower positive charge on its H atom. Consequently, HBT exhibits weaker bonding strength with specific metal sites and greater difficulty in forming hydrogen bonds with non-specific site S atoms on sulfide mineral surfaces compared to MBT, ultimately resulting in weaker adsorption capability but stronger selectivity. In summary, HBT is highly effective collector for Cu-Zn-Fe sulfide minerals and the strategy of regulating collector selectivity through electronegativity modulation has a significant potential for the development of novel highly selective collectors.