The aim of the present work was to compare and estimate absorption and biotransformation of selected selenium compounds by studying their fluxes across Caco-2 cells. Five different selenium compounds, selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), selenate, selenite, and methylseleninic acid (MeSeA), were applied to Caco-2 cells in a concentration of 10 μM, and fluxes in both directions were studied for 2 h. Fluxes of selenite and MeSeA in the presence of excess reduced glutathione (selenite + GSH and MeSeA + GSH) and flux of MeSeA in the presence of excess cysteine (MeSeA + Cys) were also studied. Selenium absorptive and exsorptive fluxes and accumulation in cell cytosol were analyzed by means of flow injection inductively coupled plasma mass spectrometry (ICP-MS). Absorptive flux of SeMet, MeSeCys, and selenate showed values correlating to complete in vivo absorption, while selenite and MeSeA fluxes correlated to poor in vivo absorption. Speciation analysis of cell lysate and donor and receptor solutions by LC-ICP-MS showed limited transformation of all selenium compounds. Extensive transformation as well as significantly increased absorptive flux was observed when co-administering selenite with glutathione compared to administering selenite alone. These observations are possibly due to formation of selenodiglutathione (GS-Se-SG) which may be absorbed differently than selenite. Concomitant application of GSH or cysteine with MeSeA resulted in extensive transformation of MeSeA, including volatile species, whereas no significant increases in fluxes were observed. In summary, the absorption of selenite selenate and the selenoamino acids is considered complete under physiological conditions, but the absorption mechanisms and metabolism of the compounds are different.