Arsenic (As) methylation in aquatic microbes plays a major role in the biogeochemistry of As. Proto-zoa, especially the free-living freshwater species, are important players in aquatic ecological health. In this study, an arsenite (As(III)) methyltransferase, TpyArsM, was identified and characterized in a free-living protozoan, Tetrahymena pyriformis. In order to confirm its function, TpyarsM gene was knocked-out in Tetrahymena and was also heterologously expressed in hypersensitive E.coli;these events resulted in expected decreases in As tolerance and methylation ability, respectively. In-vitro tests revealed that puri-fied TpyArsM protein methylated inorganic As to mono- and di- methylarsenate, and also had the novel property of producing trimethylarsenite (TMA(III)) and dimethylarsine (Me₂AsH) gases. This new methyl-transferase gene, identified in a species near the base of the food web, has enriched our knowledge of As methyltransferases and has great potential for bioremediation of As-contaminated environments.