In the branchial mitochondrion-rich (MR) cells of euryhaline teleosts, the Na(+)/K(+)/2Cl(-) cotransporter (NKCC) is an important membrane protein that maintains the internal Cl(-) concentration, and the branchial Na(+)/K(+)-ATPase (NKA) is crucial for providing the driving force for many other ion-transporting systems. Hence this study used the sailfin molly (Poecilia latipinna), an introduced aquarium fish in Taiwan, to reveal that the potential roles of NKCC and NKA in sailfin molly were correlated to fish survival rates upon salinity challenge. Higher levels of branchial NKCC were found in seawater (SW)-acclimated sailfin molly compared to freshwater (FW)-acclimated individuals. Transfer of the sailfin molly from SW to FW revealed that the expression of the NKCC and NKA proteins in the gills was retained over 7 days in order to maintain hypoosmoregulatory endurance. Meanwhile, their survival rates after transfer to SW varied with the duration of FW-exposure and decreased significantly when the SW-acclimated individuals were acclimated to FW for 21 days. Double immunofluorescence staining showed that in SW-acclimated sailfin molly, NKCC signals were expressed on the basolateral membrane of MR cells, whereas in FW-acclimated molly, they were expressed on the apical membrane. This study illustrated the correlation between the gradual reductions in expression of branchial NKCC and NKA (i.e., the hypoosmoregulatory endurance) and decreasing survival rates after hyperosmotic challenge in sailfin molly.