Bacillus sp. CDB3 isolated from an arsenic contaminated cattle dip site possesses an uncommon arsenic resistance (ars) operon bearing eight genes in the order of arsRYCDATorf7orf8. We investigated the functions of arsA, arsT, orf7 and orf8 in arsenic resistance using a plasmid-based gene knockout approach in the ars gene deficient Escherichia coli strain AW3110. The CDB3 arsA gene was shown to play a significant role in resistance, suggesting that the encoded ArsA may couple with the arsenite transporter, forming an ArsAY complex that can enhance arsenite extrusion efficiency. The disruption of either arsT or orf7 was not observed to affect arsenic resistance in the heterologous E. coli host, but their involvement in arsenic resistance can not be excluded. The orf8 gene is predicted to encode a putative dual-specificity protein phosphatase which also shares certain homology to arsenate reductases. The function loss of orf8 resulted in a remarkable decrease in resistance to arsenate, though not arsenite. To examine if this effect was due to the reduction of arsenate by orf8, the arsC gene within the 8-gene operon was disrupted. The resulting abolishment of arsenate resistance suggests that the involvement of orf8 in arsenic resistance is not via reductase activity.