Selenium (Se) shows biologically ambivalent characteristics in animals. It is an essential element but becomes severely toxic when the amount ingested exceeds the adequate intake level. Its biological, nutritional, and toxicological effects are strongly dependent on its chemical form. In this study, we evaluated the toxicity and bioavailability of nine naturally occurring Se compounds, or the so-called bioselenocompounds, in vivo and in vitro. Selenite and selenocystine showed higher toxicity than the other bioselenocompounds in vitro. In an in vitro membrane permeability study using Caco-2 cells, selenomethionine and Se-methylselenocysteine were more efficiently transported than the other bioselenocompounds. The effect of bioselenocompounds on nutritional availability was quantitatively determined from the recovery of serum selenoproteins in Se-deficient rats by speciation analysis. In contrast to the in vitro study, there were no significant differences in the assimilation of Se into serum selenoproteins among the bioselenocompounds, including selenoamino acids, selenosugar, and inorganic Se species, such as selenite, selenate, and selenocyanate, except trimethylselenonium ion. These results indicate that animals can equally assimilate both inorganic and organic naturally occurring selenocompounds except trimethylselenonium ion, which is the urinary metabolite of excess Se. We confirmed that the bioselenocompounds except trimethylselenonium ion had equivalent nutritional availabilities.