Fish are selenium rich foodstuffs and a major selenium source for the Japanese population. Niboshi is processed from Japanese anchovy (Engraulis japonicus) and commonly used to prepare soup stock for Japanese dishes. In this study, we characterized selenium species in the Niboshi extract by ultrafiltration, ion-exchange chromatography and mass spectrometry. Selenium species in the Niboshi were more extractable by polar solvents (water and ethanol) than an apolar one (hexane) along with amino acids and proteinous species. Selenium in the water-extract from the Niboshi was mostly ascribed to organoselenium compounds with a molecular mass less than 5 kDa. Although selenoamino acids and selenoproteins and their fragments were involved in the extract, a large portion of the selenium species appeared to be low-molecular-mass organoselenium compounds other than selenoamino acids and their derivatives. Ion-exchange chromatographic separations revealed that most of the selenium species in the extract possess anionic and/or amphoteric characteristics. One of these selenium species from the Niboshi extract was detected at m/z 577 for 80Se by mass spectrometry subsequent to ion-pair extraction.