The biological reduction of soluble U(VI) complexes to form immobile U(IV) species has been proposed to remediate contaminated sites. It is well established that multiheme c-type cytochromes (MHCs) are key mediators of electron transfer to aqueous phase U(VI) complexes for bacteria such as Shewanella oneidensis MR-1. Recent studies have onfirmed that the reduction proceeds via a first electron transfer forming pentavalent U(V) species that readily disproportionates. However, in the presence of the stabilizing aminocarboxylate ligand, dpaea2− (dpaeaH2bis(pyridyl-6-methyl-2-carboxylate)-ethylamine), biologically produced U(V) persisted in aqueous solution at pH 7. We aim to pinpoint the role of MHC in the reduction of U(V)-dpaea and to establish the mechanism of solid-phase U(VI)-dpaea reduction. To that end, we investigated U-dpaea reduction by two deletion mutants of S. oneidensis MR-1−one lacking outer membrane MHCs and the other lacking all outer membrane MHCs and a transmembrane MHC−and by the purified outer membrane MHC, MtrC. Our results suggest that solid-phase U(VI)-dpaea is reduced primarily by outer membrane MHCs. Additionally, MtrC can directly transfer electrons to U(V)-dpaea to form U(IV) species but is not strictly necessary, underscoring the primary involvement of outer membrane MHCs in the reduction of this pentavalent U species but not excluding that of periplasmic MHCs.