Elucidation of complex biogeochemical processes and their effects on speciation of U in the subsurface is critical for developing remediation strategies with an understanding of stability. We have developed static microcosms that are similar to bioreduction process studies in situ under laminar flow conditions or in sediment pores. Uranium L(3)-edge X-ray absorption near-edge spectroscopy analysis with depth in the microcosms indicated that transformation of U(VI) to U(IV) occurred by at least two distinct processes. Extended X-ray absorption fine structure (EXAFS) analysis indicated that initial U(VI) species associated with C- and P-containing ligands were transformed to U(IV) in the form of uraninite and U associated with Fe-bound ligands. Microbial community analysis identified putative Fe(III) and sulfate reducers at two different depths in the microcosms. The slow reduction of U(VI) to U(IV) may contribute the stability of U(IV) within microcosms at 11 months after a decrease in bioreducing conditions due to limited electron donors.