Scylla paramamosain is a euryhaline marine crab that can actively regulate its osmotic pressure of the hemolymph. We investigated the expression of Na,K-ATPase in the osmoregulatory organs, including anterior gills, posterior gills, antennal glands, and midgut. Our cDNA sequencing of the Na,K-ATPase alpha-subunit revealed no isoforms in any of the four osmoregulatory organs studied. The full-length cDNA contains 3,866 bp that encode a complete open reading frame of 1,039 amino acids. The osmotic pressure of the hemolymph changed immediately after transference from 25 ppt to 5 and 45 ppt, and reached a new steady state within 3 days, showing the typical pattern of weak hyperosmoregulators. Expression of Na,K-ATPase alpha-subunit mRNA level was determined for individuals acclimated in 5, 25, and 45 parts per thousand (ppt) for 7 days. Compared to the mRNA level of those from 25 ppt, the level in the posterior gills from 5 ppt was significantly higher. Also, the mRNA levels in the anterior gills, posterior gills and antennal glands from 45 ppt were significantly higher than that of the respective organs in 25 ppt. This increase in the 5 ppt treatment may be responsible for the observed increase of Na,K-ATPase activity on day 14 when the posterior gills had a significantly higher activity than those in the other two salinity treatments. No difference was found in the protein level across salinities. In conclusion, the posterior gills play the most important role in osmoregulation in dilute seawater and the increase in Na,K-ATPase activity on the 7th day may be due to its gene transcription and/or mRNA translation.