Elemental selenium, an essential element for humans and animals is totally obtained from foods and used for the synthesis of selenoproteins, such as glutathione peroxidases (GPx). Fish and shellfish are selenium-rich foodstuffs, and are the major dietary source of selenium for the Japanese population. However, the chemical structure and bioavailability of selenium species from seafood materials have hardly been elucidated. The Japanese littleneck clam (Asari) is the most popular consumed seawater bivalve in Japan. In this study, the selenium species in Asari were separated and assessed as a nutritional selenium source using cultured cells. The selenium content in the lyophilized Asari edible meat was 4.34±0.49 μg/g. The selenium extraction rate into water was 17−24% (1.07±0.14 μg/g-dry Asari). Based on the results that selenium in the Asari water-extract was mostly retained on both cationic Q Sepharose and anionic SP Sepharose columns, the selenium species in the Asari water-extract appeared to possess an amphoteric character. Selenium in the Asari water-extract mostly passed through a membrane with the molecular mass cutoff of 5000. After lyophilization, the resulting filtrate was subjected to in vitro assessment of the selenium bioavailability. HepG2 and HeLa cells were cultured in a basal medium containing the filtrate. The selenium content and GPx activity of the HepG2 cells increased with the increasing selenium content in the medium and incubation time, which suggested that the selenium species in the Asari water-extract were utilized for the synthesis of the GPxs. Overall, these results demonstrated that Asari contains nutritionally effective selenium species.