Major uranium (U) deposits worldwide are exploited by acid leaching, known as 'in-situ recovery' (ISR). ISR involves the injection of an acid fluid into ore-bearing aquifers and the pumping of the resulting metal-containing solution through cation exchange columns for the recovery of dissolved U. Rehabilitation of ISR-impacted aquifers could be achieved through natural attenuation, or via biostimulation of autochthonous heterotrophic microorganisms due to the associated acid neutralization and trace metal immobilization. In this study, we analyzed the capacity of pristine aquifer sediments impacted by diluted ISR fluids to buffer pH and immobilize U. The experimental setup consisted of glass columns, filled with sediment from a U ore-bearing aquifer, through which diluted ISR fluids were flowed continuously. The ISR solution was obtained from ISR mining operations at the Muyunkum and Tortkuduk deposits in Kazakhstan. Following this initial phase, columns were biostimulated with a mix of molasses, yeast extract and glycerol to stimulate the growth of autochthonous heterotrophic communities. Experimental results showed that this amendment efficiently promoted the activity of acid-tolerant bacterial guilds, with pH values rising from 4.8 to 6.5 - 7.0 at the outlet of the stimulated columns. The reduction of sulfate, nitrate, and metals as well as dissimilatory nitrate reduction to ammonia induced the rise in pH values, in agreement with geochemical modeling results. Biostimulation efficiently promoted the complete immobilization of U, with the accumulation of up to 3,343 ppm in the first few centimeters of the columns. Synchrotron analysis and SEM-EDS revealed that up to 60% of the injected hexavalent U was immobilized as tetravalent non-crystalline U onto bacterial cell surfaces. 16S rDNA amplicon analysis and qPCR data suggested a predominant role played for members of the Phylum Firmicutes (from the genera Clostridium, Pelosinus and Desulfosporosinus) in biological U reduction and immobilization.