As-contaminated soil was considered to be harmful, while low-As soil was often neglected in health risk assessment. Considering the pathopoiesia of As at low concentration and noticeable ingestion of low-As soil, this research investigated the As bioaccessibility and species of low-As soil in whole digestive tract and the role of colon microbes in the regulation of As bioaccessibility and its species by utilizing a combined in vitro model UBM-SHIME (Unified BARGE Method-Simulator of Human Intestinal Microbial Ecosystem). Results indicated that the As bioaccessibility of all the soils to range from 11.39% to 20.99% in stomach, from 12.42% to 34.48% in small intestine and from 11.75% to 22.92% in colon, respectively. Speciation analysis showed only inorganic arsenic (iAs) was detected in stomach and the small intestinal extraction solution, with arsenate (As(V)) being the dominated As speciation (70.5%~87.9% of total As in extraction solution). In addition, As species was significantly altered by the active colon which resulted in the formation of methylated As species. Except for monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)) of low toxicity, a highly toxic As species, monomethylarsonous acid (MMA(III)), was observed in the colon. Moreover, a high level of monomethylmonothioarsonic acid (MMMTA(V)) (up to 25.9% of total As in extraction solution ) of unclear toxicological properties was also detected. The formation of various organic As species demonstrated that human colon microorganisms had the ability to actively metabolise iAs into methylated arsenicals and methylated thio-arsenicals. Such transformation should be considered seriously when assessing the human health risks associated with oral exposure to low-As soil.