Changes in mRNA levels of prolactin (PRL) during upstream movement were examined in juvenile Japanese eels, Anguilla japonica. Glass eels and elvers were collected from 2007 to 2009 near the entrance of Hamana Lake, and in a small inflowing stream, the Egawa River. Quantification of mRNA was performed by real-time PCR and expressed as whole-body content. PRL mRNA levels of glass eels caught in the coastal zone and tidal area were low. Eels that moved downward in the tidal zone and migrated upstream to enter into freshwater showed increased levels of PRL mRNA. These changes suggest the importance of up-regulation of PRL gene expression in juvenile eels during their upstream movement from seawater to fresh water, particularly in relation to hyperosmoregulation.