Upon hyperosmotic or hypoosmotic challenge, Na+/K+-ATPase (NKA) in gills of the teleosts supplied driving force for ion-secreting or ion-absorbing mechanisms, respectively. Meanwhile, FXYD11 plays important roles in modulating (NKA) activity in gills of the teleosts. Little is known, however, about the details of interaction between FXYD11 and NKA upon acute salinity challenges. The brackish medaka is a euryhaline teleost and a useful experimental animal model, which are able to tolerate a broad range of environmental salinities with efficient osmoregulatory mechanisms to maintain homeostasis. Time-course alterations in branchial FXYD11 and NKA expression (mRNA/protein), as well as NKA activity after exposure to salinity changes were first examined in this study to realize the short-term effects of different salinity stresses on FXYD11 regulated NKA activity in gills of the brackish medaka. The brackish medaka were acclimated to brackish water (BW, 15‰) for at least four weeks, and then the BW-acclimated medaka were transferred directly to fresh water (FW) or seawater (SW, 35‰). Upon hyperosmotic and hypoosmotic challenge, the NKA activity significantly increased at 1 hr (7.09 ± 0.80 vs. 5.50 ± 0.96 umol of ADP / mg protein / hr, p<0.05) and 12 (7.92 ± 1.05 vs. 5.50 ± 0.96 umol of ADP / mg protein / hr, p<0.05) hrs, respectively, while no significant difference of NKA protein abundance was found compared with the control group. Moreover, there was no significant difference found in the levels of FXYD11 mRNA in the salinity stresses. When the medaka were transferred from BW to FW, the FXYD11 protein abundance significantly increased since 12 hrs. Meanwhile, no significant difference was found in the FXYD protein abundance when transferred from BW to SW. Therefore, at 12 hrs upon hyperosmotic challenge, the branchial NKA activity and FYXD11 protein amounts were up-regulated, but NKA protein amounts did not change significantly. On the other hand, at 1 hr upon hypoosmotic challenge, the NKA activity was up-regulated, but NKA and FXYD11 protein amounts were not significantly different. The co-immunoprecipitation results further illustrated that the interaction between NKA and FYXD11 was up-regulated at 12 hrs upon hyperosmotic challenge, but not significantly different at 1 hr upon hypoosmotic challenge. The present study suggested that the brackish medaka used different mechanisms to modulate NKA activity when acclimated to hypoosmotic or hyperosmotic environments. FXYD11 might regulate NKA activity efficiently upon hyperosmotic challenge.