Rice is the primary source of arsenic exposure to humans. Changes in arsenic behavior within paddy soil influence its accumulation in rice. In this study, we investigated how the simultaneous application of rice straw and sulfate affects arsenic behavior. We observed arsenic mobility and speciation under two repetitive reduction (20 days) and oxidation (28 days) conditions after applying rice straw (1% w/w) and sulfate (280 mg/L) through microcosm experiments. Changes in arsenic and iron forms were determined using Wenzel sequential extraction and X-ray absorption near-edge structure-linear combination fitting (XANES-LCF). The co-application of rice straw and sulfate (As+RS+S) resulted in the highest total arsenic concentration (20.7 mg/L) in pore water during the first reduction-oxidation cycle. This was presumably due to the increased reductive dissolution following rice straw application and the increased abundance of iron and sulfate reducing microorganisms after co-application. As+RS+S showed the highest pyrite abundance in the first reduction-oxidation cycle. The elevated arsenic mobility in As+RS+S was possibly a result of competition between arsenic and dissolved organic carbon in the pore water. Also, the formation of dimethylated arsenicals (dimethylarsinic acid (DMA), dimethylmonothioarsinic acid (DMMTA), dimethyldithioarsinic acid (DMDTA)) in paddy soil pore water was confirmed during the first reduction-oxidation cycle when rice straw and sulfate were added. This is presumed to be due to the promotion of reducing conditions by the application of rice straw and sulfate, as well as the increased abundance of sulfate reducing microorganisms. On the other hand, the total arsenic concentration decreased by about 87% in the second reduction-oxidation cycle, possibly due to the reduction of dissolved organic carbon and increased iron oxide crystallinity.