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Bioavailability and speciation of arsenic (As) are impacted by fertilization and bacteria in the rice rhizosphere. In this study, we investigated the effects of long-term manure application on As bioavailability, microbial community structure, and functional genes in a rice paddy field. The results showed that manure application did not affect total As in the soil but increased soluble As forms by 19%, increasing arsenite (As­(III)) accumulation in rice grains and roots by 34 and 64% compared to a control. A real-time quantitative polymerase chain reaction (qPCR) and high-throughput sequencing analysis demonstrated that manure application increased the relative abundance of Rhizobium, Burkholderia, Sphingobium, and Sphingomonas containing arsenate reductase genes (arsC) in the rhizosphere soil, consistent with the 529% increase in arsC, which may have promoted arsenate (As­(V)) reduction and increased As availability in pore water. In addition, manure application significantly altered the iron (Fe)-plaque microbial community structure and diversity. The microbes, particularly, Bradyrhizobium, Burkholderia, and Ralstonia, were mostly associated with As, Fe, and sulfur (S) cycles. This result was consistent with changes in the functional genes related to As, Fe, and S transformation. Although manure application promoted As­(V) reduction (arsC) in Fe-plaque by 682%, it inhibited Fe and S reduction by decreasing FeIII reduction bacteria (Geobacteraceae) and the sulfate-reducing gene (dsrA) abundance. Further, manure application changed the composition of the microbial community that contained the arsC gene. In short, caution needs to be excised even in the soil with a low As concentration as manure application increased As­(III) accumulation in rice grains.