The lime-depressed pyrite from Cu differential flotation tailings with acid mine drainage (AMD) as a natural activator was recovered. The effect of AMD on lime-depressed pyrite flotation was investigated by a series of laboratory flotation tests and surface analytical techniques. Flotation test results indicated that AMD could effectively activate the pyrite flotation with a sodium butyl xanthate (SBX) collector, and a high-quality sulfur concentrate was obtained. Pulp ion concentration analysis results indicated that AMD facilitated desorption of Ca²⁺ and adsorption of Cu²⁺ on the depressed-pyrite surface. Adsorption measurements and contact angle analysis results confirmed that adding AMD improved the adsorption amount of SBX collector on the pyrite surface and increased the contact angle by 31°. Results of Raman spectroscopy and X-ray photoelectron spectroscopy analysis indicated that AMD treatment promoted the formation of hydrophobic species (S⁰ hydrophobic entity and copper sulfides) and the removal of hydrophilic calcium and iron species on the pyrite surface, which reinforced the adsorption of collector. The findings of the present research provide important theoretical basis and technical support for a cleaner production of copper sulfide ores.