To understand selenium (Se) circulation in the biosphere, the metabolism of organic Se, in particular, Se metabolites, in animals and plants should be elucidated. In this study, garlic, Allium sativum, a well-known Se accumulator with high Se metabolic ability, was hydroponically cultivated and then exposed to trimethylselenonium ion (TMSe), a urinary metabolite. Thereafter, the Se concentration in several parts of garlic, such as roots, bulbs, and leaves, was determined. To reveal the metabolic pathway of TMSe, the Se species in A. sativum were investigated by speciation using HPLC hyphenated with an inductively coupled plasma mass spectrometer (LC-ICP-MS). Se was mostly accumulated in the roots. TMSe was detected in the extract of each plant part. However, the amount of Se incorporated from the medium was not completely recovered in the garlic, suggesting that a part of TMSe was metabolized into volatile Se. Consequently, we conclude that the majority of TMSe incorporated into garlic is accumulated as is, the rest is partially desmethylated to form a volatile Se compound, such as a dimethylated Se compound.