The onset of osmoregulation for seawater adaptation was examined during the early life stages of the Japanese eel. Ingested seawater was detected in the digestive tract by using fluorescent dextran as an inert marker. Ingested seawater remained in the forepart of the digestive tract at 0 and 1 days post hatching (dph), but reached the anus at 2 dph. Scanning electron microscope observations showed that the mouth appeared as a slit at 0 dph and developed into a hole-like shape at 2 dph. Expressions of Na + , K + , and 2Cl − cotransporter 2β (NKCC2β), and Na + and Cl − cotransporter β (NCCβ) mRNAs were detected mainly in the intestine and rectum, respectively. These results are consistent with those of the adult eel, suggesting that the intestine and rectum are the sites of active ion absorption in larvae also. Expression levels of NKCC2β steeply increased from 4 to 6 dph, while NCCβ levels were highest on the day of hatching, presumably due to a maternal factor. The expression levels of NCCβ decreased by 3–4 dph and then increased to a constant status at 7 dph. Our findings confirmed that osmoregulation started as early as the day of hatching and stabilized within a week.