The bioaccessibility of arsenic and its speciation are two important factors in assessing human health risks exposure to contaminated soils. However, the effects of human gut microbiota on arsenic bioaccessibility and its speciation are not well characterized. In this study, an improved in vitro model was utilized to investigate the bioaccessibility of arsenic in the digestive tract and the role of human gut microbiota in the regulation of arsenic speciation. For all soils, arsenic bioaccessibility from the combined in vitro model showed that it was <40% in the gastric, small intestinal and colon phases. This finding demonstrated that the common bioaccessibility approach assuming 100% bioaccessibility would overestimate the human health risks posed by contaminated soils. Further to this, the study showed that arsenic bioaccessibility was 22% higher in the active colon phase than that in the sterile colon phase indicating that human colon microorganisms could induce arsenic release from the solid phase. Only inorganic arsenic was detected in the gastric and small intestinal phases, with arsenate [As(V)] being the dominant arsenic species (74%–87% of total arsenic). Arsenic speciation was significantly altered by the active colon microbiota, which resulted in the formation of methylated arsenic species, including monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] with low toxicity, and a highly toxic arsenic species monomethylarsonous acid [MMA(III)]. Additionally, a high level of monomethylmonothioarsonic acid [MMMTA(V)] (up to 17% of total arsenic in the extraction solution) with unknown toxicological properties was also detected in the active colon phase. The formation of various organic arsenic species demonstrated that human colon microorganisms could actively metabolize inorganic arsenic into methylated arsenicals and methylated thioarsenicals. Such transformation should be considered when assessing the human health risks associated with oral exposure to soil.