Neutral in-situ leaching (ISL) is a prominent technique for extracting uranium (U) from sandstone-type U deposits. However, the intricate biogeochemical processes and associated microbial communities in environments subjected to prolonged neutral in-situ U leaching remain insufficiently understood. Here, capitalizing on groundwater samples collected from an area subjected to two decades of ISL (Undergoing-ISL) and a background aquifer (Pre-ISL) at the Qianjiadian U mining site (the first CO2+O2 ISL site located in the Songliao Basin of northern China) we investigate the responses of aquifer microbiomes to CO2+O2 ISL using genome-resolved metagenomics resulting in a total of 315 good-quality bacterial metagenome-assembled genomes (MAGs) and 5 archaeal genomes. Five putative keystone MAGs were identified based on their high connection degree and low betweenness centrality within the co-occurrence network of reconstructed MAGs, notably highlighting the prevalence of microorganisms involved in sulfur, ammonium, and Fe(Ⅱ) oxidation, which are integral to facilitating U(Ⅳ) oxidation during CO2+O2 ISL. In particular, sulfur-oxidizing bacteria and Fe(Ⅱ)-oxidizing bacteria (Rhodoferax and SHZL01) played a key role in U mobilization during long-term neutral ISL. These findings enhance our understanding of subsurface microbial ecology and hold significant implications for bioremediation strategies and groundwater management in regions affected by mining.