Microbial reduction of U(VI) is an important phenomenon affecting uranium mobility in the subsurface environments. Elucidation of its mechanism is necessary for predicting uranium migration and for applying environmental remediation. In this study, we have examined the electron pathway for the U(VI) reduction mediated by flavin mononucleotide (FMN), which is secreted by Shewanella species. The cyclic voltammetry (CV) and photo-electrochemical methods with an optically transparent thin-layer electrode (OTTLE) cell were utilized in investigating in vitro the electron transfer reactions that take place between FMN and U(VI). The CV measurements of U(VI) were carried out in a citrate and Tris-HCl buffer both with and without FMN. A scarce U(VI) reduction current was observed in the absence of the FMN. To the contrary, a catalytic U(VI) reduction current was observed in the presence of FMN at the redox potential of the FMN. The reduction current increased with an increase in the concentration of the U(VI). The reduced form of the U was confirmed to be U(VI) by the photo-electrochemical analysis using the OTTLE cell. The results demonstrated that FMN acts as a mediator in the electro-reduction of U(VI) to U(iv). In addition, in vivo bio-reduction experiments on U(VI) with Shewanella putrefaciens revealed that the addition of FMN accelerated the reduction rate of U(VI). These results indicate that the bio-reduction of U(VI) by the Shewanella species can be catalyzed by FMN secreted from the cells.