Abstract: The gills are the major apparatus for osmoregulation in fish to acclimate the changes of salinities. Na⁺/K⁺/2Cl⁻cotransporter 1-α (NKCC1α) is one of the key ion cotransporter locoalized in gill chloride cells which has been associated with the maintence of osmotic homeostasis. The transport process mediated by NKCC1α is characterized by electroneutrality with a stoichiometry of 1Na:1K:2Cl. Sarotherodon melanotheron is one of the most euryhaline teleosts able to withstand variations in environmental salinity ranging from freshwater to hyper-saline waters. In this study, the reverse transcription-polymerase chain reaction and rapid amplification of 3' and 5'cDNA ends methods were used to identify the full cDNA of the NKCC1α with an Open Reading Frame which contains 1 151aa of S.melanotheron. The amino acid multiple alignment and phylogenetic analysis showed that this isoform is more similar with isoforms in Oreochromis mossambicus, Salmo salar and Anguilla anguilla, and there is the highest homologous of 99% between Sarotherodon and Mossambique. The predicted protein secondly structure of NKCC1α contains 10 transmenbrane domains, which were highly conserved in sequences and locoalization sites relatively to other species. The quantitative real time polymerase chain reaction (qRT-PCR) assay was developed to estimate the mRNA expression levels in gill, liver, intestine and kidney in freshwater, the results showed a tissue-specific model. Furthermore, the sanility significantly affects the relative expression level of NKCC1α mRNA in gill with a 4.9 times higher in 136 salinity water than that in 0 salinity. The results suggest that the NKCC1α is closely related to the salt tolerance in S.melanotheron.