The natural enrichment of arsenic (As) in the shallow groundwater of the Jianghan Plain, central Yangtze River Basin, has been reported recently. However, the mechanism of As mobilization in the aquifer is not clearly understood yet. To evaluate the effect of microbial sulfate-reducing activities on As mobilization in the aquifer, the sediment samples were collected from 20.75 m at the typical As-rich depth of shallow aquifer. The results indicated that 3 mg kg−1 As released from the sediment with acetate amended or without amendment. It was notable that, in the late cultural period, the Fe(II) was scavenged in the aqueous phase along with the decrease of sulfate, suggesting that the Fe-sulfide mineral may be generated in the incubation systems. Correspondingly, high abundance of sulfate-reducing bacteria Desulfomicrobium were observed, indicating the occurrence of bacteria sulfate reduction. Those results suggested that the As was mobilized under the sulfate reducing condition, and furthermore, the released As was not immobilized by the re-generation of Fe-sulfide minerals. These results can provide new insights for the mobilization mechanism of As in aquifer systems.