Previously, we found that arsenite (As-III) oxidation could improve the generation of ATP/NADH to support the growth of Agrobacterium tumefaciens GW4. In this study, we found that aioE is induced by As-III and located in the arsenic island near the As-III oxidase genes aioBA and co-transcripted with the arsenic resistant genes arsR1-arsC1-arsC2-acr3-1. AioE belongs to TrkA family corresponding the electron transport function with the generation of NADH and H+. An aioE in-frame deletion strain showed a null As-III oxidation and a reduced As-III resistance, while a cytC mutant only reduced As-III oxidation efficiency. With As-III, aioE was directly related to the increase of NADH, while cytC was essential for ATP generation. In addition, cyclic voltammetry analysis showed that the redox potential (ORP) of AioBA and AioE were + 0.297 mV vs. NHE and + 0.255 mV vs. NHE, respectively. The ORP gradient is AioBA > AioE > CytC (+ 0.217 similar to + 0.251 mV vs. NHE), which infers that electron may transfer from AioBA to CytC via AioE. The results indicate that AioE may act as a novel As-III oxidation electron transporter associated with NADH generation. Since As-III oxidation contributes As-III detoxification, the essential of AioE for As-III resistance is also reasonable.