Abstract: Acid mine drainage (AMD) poses a serious threat to the ecological environment due to its strong acidity, high sulfate, and heavy metal content. Sulfate-reducing bacteria (SRB) offer a promising bioremediation strategy, yet their enrichment behavior and adaptation to varying initial pH conditions remain insufficiently understood. This study systematically investigated the effects of initial pH values on SRB enrichment characteristics and wastewater treatment efficiency. Indigenous SRB were enriched in Postgate medium with gradient pH (3.0, 4.0, 5.0, 6.0, 7.0) on AMD substrate from a coal mine in Chongqing. The results show that the initial pH significantly affected the enrichment efficiency of SRB. Enrichment was unsuccessful at pH 3.0−4.0, delayed at pH 5.0 (25 days), and most efficient at pH 6.0−7.0 (12 days). Under favorable conditions (pH 5.0−7.0), SRB activity increased system pH to 6.7−7.0 and reduced redox potential from 267.8 mV to −400.0 mV, confirming strong reducing capacity. At pH 6.0−7.0, SRB removed 100.0% of Fe²⁺ with in 44 days, and 67.9%～72.6% of sulfate (446.6 mg/L−522.7 mg/L), while soluble sulfide concentrations rose to 96.6～102.9 mg/L. Delayed but comparable removal efficiencies were observed at pH 5.0. Microbial community analysis indicates that SRB were successfully enriched at an initial pH of 5.0−7.0. The dominant SRB genus was Desulfosporosinus (relative abundance 11.0%−34.5%), which was most active at pH 6.0. Indigenous strains (CQ@pH5, CQ@pH6, and CQ@pH7) grew best under neutral conditions (pH 6.0−7.0), had the highest rate of sulfate reduction (soluble sulfide yield up to 38.8 mg/L in 12 days), and 100% Fe²⁺ removal, while the extreme acidic environment (pH≤4.0) completely inhibited their activity. This study elucidates the pH-dependent enrichment and functional mechanisms of indigenous SRB, providing a theoretical basis and practical foundation for the optimization of SRB-driven AMD remediation technologies.