Oral intake of purified selenoneine and seafoods has been reported to result in selenoneine accumulation in erythrocytes in mice and human. In addition, Se-methylselenoneine was suggested to be produced as a metabolite of selenoneine in the urine and whole blood of humans. In order to confirm the molecular mechanism of production of Se-methylselenoneine, a stable isotope (Se-76) labeled selenoneine was biosynthesized using genetically modified fission yeast and administered to mice. The Se-76-labeled Se-methylselenoneine was detected in urine but Se-78 and Se-80-labeled Se-methylselenoneine arising from natural isotopes of Se was hardly detected. These results suggest that Se-methylselenoneine was a metabolite and the excreted form of selenoneine. The methylation of selenoneine in mice administered selenoneine continuously was evaluated by the analyses of organs using an online liquid chromatograph system with an inductively coupled plasma mass spectrometer (LC-ICP-MS). These experiments indicate that selenoneine is methylated in the liver and (or) kidneys.