Nitrate and arsenic are common pollutants in groundwater. However, we are still lacking technologies that can remove nitrate and arsenic simultaneously using a single species of microorganism. In the present study, the potential of synchronous removal of nitrate and arsenite from water by a single anaerobic nitrate-reducing Fe(II) oxidizing Citrobacter freundii strain PXL1 was assessed. The strain PXL1 could grow well in arsenite concentration of 2.85–13.65 μM and efficiently oxidize Fe(II), reduce nitrate and remove As(III) from water. Scanning electron microscopy, X-ray diffraction spectroscopy, and Fourier transform infrared spectroscopy analysis showed that the strain PXL1 produced poorly crystalline Fe(III) oxides. As(III) was removed mainly by adsorption and co-precipitation of the biogenic Fe(III) oxides. The anaerobic nitrate-dependent Fe(II) oxidizing bacteria including C. freundii PXL1 are promising microbes for in situ remediation of nitrate and arsenite contaminated groundwater.