Decades of uranium-related activities such as ore mining, nuclear power generation, weapon manufacture, storage of nuclear wastes, have contributed to the release of U in the environment. U occurrence is concerning, since in surface environments, U is typically found in the U(VI) oxidation state, as uranyl, complexed with various ligands, and is highly soluble. Therefore, this toxic metal is likely to leech into soils and contaminate surface waters and groundwaters. In addition, owing to the long half-lives of the predominant isotopes 238U and 235U, U is considered as a persistent contaminant over geological timescales. For several decades, efforts have focused on the development of in situ remediation solutions to tackle U pollution in the subsurface, and limit its mobility. With this aim, interest in the metabolic potential for U(VI)-respiration by dissimilatory metal-reducing bacteria (DMRB), such as Shewanella oneidensis MR-1, has seen a significant increase. This metabolically-versatile bacterium has been reported to reduce mobile U(VI) to typically insoluble crystalline and amorphous U(IV). The reduction of U(VI) in S. oneidensis MR-1 is coupled to the oxidation of an electron donor, which feeds electrons into an electron transport chain, extending from the cytoplasm to the outer-membrane of the cells. The microbially mediated reduction of U(VI) to U(IV) is the result of a two-step process. It is assumed that one electron is first transferred to U(VI) to form a pentavalent U(V) intermediate, followed by the abiotic disproportionation of two U(V) atoms into U(IV) and U(VI). However, evidence for this mechanism is limited to experimental systems rich in carbonate, which permits the rapid disproportionation of U(V). Thus, it remains unclear whether a second, biologically-mediated, electron transfer to U(V) is possible under conditions in which disproportionation is limited. To explore this, a novel U(V)-dpaea complex, that is stable in water at pH 7, was utilized to investigate the second step of the reduction mechanism. Here, we observed that U(V) can be biologically reduced by an additional one-electron transfer, resulting in the accumulation of U(IV) without the need for disproportionation. To improve our understanding of the molecular mechanism of U-dpaea reduction, we incubated mutant strains of S. oneidensis MR-1, lacking (i) only outer-membrane c-type cytochromes or (ii) all c-type cytochromes, with solid phase U(VI)-dpaea and aqueous U(V)-dpaea. We determined that U(VI)-dpaea reduction proceeds via the initial dissolution of the solid phase and that U(V)-dpaea reduction is mediated by outer-membrane c-type cytochromes. In particular, in vitro reactions between the purified outer-membrane c-type cytochrome MtrC and U(V)-dpaea demonstrated that MtrC can directly transfer electrons to U(V)-dpaea. Finally, we sought to determine the factors that influence electron transfer kinetics between DMRB and U. To this end, we reacted U(VI) coordinated by various aminocarboxylate ligands with purified MtrC. Here, U speciation significantly impacted reduction rates and appeared to be related to the binding strength of the U-MtrC interaction, i.e., hydrogen bonding versus electrostatic. All together, these findings provide further insights in the reduction mechanism of U by DMRB, and underline the importance of U speciation in controlling the pathway and rate of electron transfer.