||Euryhaline milkfish (Chanos chanos) is an important economic species in Taiwan and is able to survive in both seawater (SW) and fresh water (FW). Upon salinity changes, euryhaline fish kidneys play a crucial role in osmoregulation. During this process, Na+/K + -ATPase (NKA) mainly functions in providing the driving force for the other ion-transporting systems. In the milkfish, NKA activities have been widely studied, yet the isoforms and modulatory mechanisms of the NKA α-subunits are unknown. Hence, this study was undertaken to reveal potential modulatory mechanisms, including the isoform composition of NKA α-subunits and FXYD proteins (the modulators of NKA), of NKA expression/activity in milkfish kidneys in response to salinity changes. Four isoforms (CcNKA α1a, α1c, α2, and α3) of NKA α-subunits with distinct tissue distribution were identified in milkfish. Among them, Ccnka α1a and Ccfxyd2 were predominantly expressed in the kidney. Upon salinity challenge, parallel expression patterns were found in renal Ccnka α1a and Ccfxyd2, showing an increase in the FW-acclimated milkfish but not in the SW-acclimated group. After transfer, no matter from SW to FW or FW to SW, most of the renal Ccnka and Ccfxyd genes (particularly Ccnka α1a and Ccfxyd2) significantly changed. Overall, these results suggest that the NKA activity of milkfish kidneys may be modulated via CcNKA α1a and CcFXYD2 for osmoregulation in response to salinity challenges.