In this study, Fe(0) and mixed anaerobic culture were integrated in one column to investigate the coupled abiotic and biotic effects on hexa-valent chromium (Cr(VI)) removal and column longevity with an abiotic Fe(0) column in the control experiments. According to the breakthrough study, a slower Cr(VI) breakthrough rate of 0.19 cm/PV was observed in the biotic Fe(0) column whereas the value in the abiotic Fe(0) column was 0.30 cm/PV, resulting in 64% longer life-span and 62% higher Cr(VI) removal capacity in the biotic Fe(0) column than the abiotic one. The solid phase characterization by scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and X-ray diffraction (XRD) confirmed that this enhancement was attributed to the higher consumption of iron and greater production of diverse reactive minerals (e.g., green rust, magnetite and lepidocrocite) induced by the synergistic interaction of Fe(0) and anaerobic culture, providing more reactive sites for Cr(VI) adsorption, reduction and co-precipitation. Furthermore, the decreasing breakthrough rates and growing iron corrosion along the biotic Fe(0) column demonstrated an inhomogeneous distribution of reactive zones in the column and its latter 3/5 section was considered to be the most reactive area for Cr(VI) removal. These results indicate that the inoculation of microorganisms in Fe(0)-based permeable reactive barriers will enable this technology a higher removal capacity and longer life-span for the remediation of Cr(VI)-contaminated groundwater.