Anthraquinone-2-sulfonate(AQS) was employed in humus substitutes to evaluate the effects and influencing factors of U(VI) reduction by Shewanella oneidensis MR-1(S. oneidensis MR-1) under anaerobic condition. The removal rate of U(VI) at 30 °C reaches 99.0% afterd 96 h with the p H value of 7.0 and AQS concentration of 1.0 mmol/L. The effective concentrations of AQS as the accelerator for U(VI) bioreduction are approximately 0.5-1.0 mmol/L. The bioreduction of U(VI) is inhibited when the concentration of AQS exceeds 2.0 mmol/L. The coexistence of ions, such as Cu2+, Cr6+, Mn2+, shows a remarkable negative effect on the U(VI) reduction, and Zn2+ shows less influence on the process compared with other tested ions. The U(VI) reduction is remarkably inhibited when the concentration of nitrate ion exceeds 1.0 mmol/L. Otherwise, no difference is found when the nitrate ion concentration is less than 0.5 mmol/L. Sulfate ion(5.0 mmol/L) slightly promotes the U(VI) reduction. Zero-valent iron(ZVI) promotes the U(VI) reduction by S. oneidensis, and the reduction rate improves with increasing the amount of ZVI in the range of 0-2.0 g/L. The XPS result indicates that uranium deposits on the cell surface are in U(VI) and U(IV) forms, and the majority of uranium in the solution is stable UO2.