Microbial U(VI) reduction influences uranium mobility in contaminated subsurface environments and can affect the disposal of high-level radioactive waste by transforming the water-soluble U(VI) to less mobile U(IV). The reduction of U(VI) by the sulfate-reducing bacterium Desulfosporosinus hippei DSM 8344^T, a close phylogenetic relative to naturally occurring microorganism present in clay rock and bentonite, was investigated. D. hippei DSM 8344^T showed a relatively fast removal of uranium from the supernatants in artificial Opalinus Clay pore water, but no removal in 30 mM bicarbonate solution. Combined speciation calculations and luminescence spectroscopic investigations showed the dependence of U(VI) reduction on the initial U(VI) species. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed uranium-containing aggregates on the cell surface and some membrane vesicles. By combining different spectroscopic techniques, including UV/Vis spectroscopy, as well as uranium M₄-edge X-ray absorption near-edge structure recorded in high-energy-resolution fluorescence-detection mode and extended X-ray absorption fine structure analysis, the partial reduction of U(VI) could be verified, whereby the formed U(IV) product has an unknown structure. Furthermore, the U M₄ HERFD-XANES showed the presence of U(V) during the process. These findings offer new insights into U(VI) reduction by sulfate-reducing bacteria and contribute to a comprehensive safety concept for a repository for high-level radioactive waste.