[Objective] Reductive biotransformation of 2,4-dinitrotoluene (2,4-DNT) by Shewanella oneidensis MR-1 was evaluated under anaerobic conditions. [Methods] Four systems were set up where 2,4-dinitrotoluene (2,4-DNT), S. oneidensis MR-1 and flavins were used as electron acceptor, degrading bacteria and extracellular mediator, respectively. Concentrations of 2,4-DNT and its metabolites were monitored by time temporal sampling. Growth of S. oneidensis MR-1 under different concentrations of 2,4-DNT and biotransformation of 2,4-DNT under different concentrations of flavins were also studied. [Results] S. oneidensis MR-1 could efficiently biotransform 2,4-DNT to 4-amino-2-nitrotoluene (4A2NT) or 2-amino-4-nitrotoluene (2A4NT), both of them were further reduced to 2,4-diaminotoluene (2,4-DAT). The presence of flavins could accelerate the biotransformation. [Conclusion] S. oneidensis MR-1 showed a high capacity for biotransforming 2,4-DNT and could be further applied in the in situ bioremediation of nitrobenzene contaminations in the environment.