During the Mining-Agriculture Complex Areas, the mining and agriculture activities could lead to an excessive presence of sulfate content in the regional groundwater. Sulfate exhibits the potential to influence the positive accumulation of RGs, although its mechanisms remain inadequately explored. To address this gap, this study analyzed the RGs buildup mechanisms in the groundwater of the mining-agriculture complex area. Results showed a widespread presence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs), especially in coal-seams crevice groundwater. And iron and sulfur are primary environmental factors conducive to RGs accumulation through a synergistic interaction. Microbial annotation of gene sets sourced from coal-seams crevice groundwater samples unveiled part of sulfur-metabolizing microorganisms that were hosts of both ARGs and MRGs. Mechanistic insights revealed that iron may stimulates reactive oxygen species (ROS) generation, facilitating RGs accumulation, while adjusting sulfur metabolism and the synthesis of iron-sulfur clusters, thereby augmenting microbial growth which as predominant hosts of RGs, thereby intensifying the buildup of RGs.