The Cr(VI) removal way and Cr(VI) reducing site of Oceanobacillus oncorhynchi W4, a novel Cr(VI) reducing bacterium, were investigated in this study. Results showed that about 74.2% of Cr(VI) was removed from solution by growing cells within 72 h. Moreover, heating-killed resting cells had little Cr(VI) removal capacity, which was significantly lower than that of resting cells, which reached nearly 80% removal rate, suggesting that the way of Cr(VI) removal mainly relied on biological reduction rather than biosorption. And the Cr(VI) reduction was found to be significantly enhanced by some electron donors, especially glycerin, which further verified enzyme-mediated biological reduction as the way for Cr(VI) removal. Experiments of Cr(VI) removal by permeable cells indicated that there was no significant difference in chromium reduction between the impermeable cells and the permeable cells. The cell envelop fraction had a Cr(VI) removal rate of 82.9%, apparently higher than cytoplasmic fraction (11.1%), indicating that the cell envelop was the main location for Cr(VI) reduction, which were further demonstrated by Scanning Electron Microscope and Transmission electron microscopy plus EDS analysis. Furthermore, analysis of X-ray photoelectron spectroscopy manifested that C=O, C-OH and C-O-C groups on the surfaces played major roles in correlation with chromium species.