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Asian sea bass (Lates calcarifer Bloch, 1790) is a euryhaline fish widely cultured in Asia and Australia. Although it is common to culture Asian sea bass at different salinities, osmoregulatory responses of Asian sea bass during acclimation to various salinities have not been fully observed. In this study, we used scanning electron microscopy to observe the morphology of the ionocyte apical membrane of Asian sea bass acclimated to fresh water (FW), 10‰ brackish water (BW10), 20‰ brackish water (BW20), and seawater (SW; 35‰). Three types of ionocytes were identified in FW and BW fish: (I) flat type with microvilli, (II) basin type with microvilli, and (III) small−hole type. Flat type I ionocytes were also observed in the lamellae of the FW fish. In contrast, two types of ionocytes were identified in SW fish: (III) small‐hole type and (IV) big‐hole type. Furthermore, we observed Na+, K+‐ATPase (NKA) immunoreactive cells in the gills, which represent the localization of ionocytes. The highest protein abundance was observed in the SW and FW groups, whereas the highest activity was observed in the SW group. In contrast, the BW10 group had the lowest protein abundance and activity. This study demonstrates the effects of osmoregulatory responses on the morphology and density of ionocytes, as well as protein abundance and activity of NKA. In this study, we found that Asian sea bass had the lowest osmoregulatory response in BW10, because the lowest amounts of ionocytes and NKA were required to maintain osmolality at this salinity. Research highlights: Three types of ionocyte apical membranes were identified in fresh and brackish water (BW) Asian sea bass, and two types in seawater fish.Na+, K+‐ATPase immunoreactive (NKIR) cells were observed in the gills, and their protein abundance and activity were highest in the seawater and freshwater groups.The lowest osmoregulatory response was found in the 10‰ BW group with the lowest amount of ionocytes and NKA required to maintain osmolality. [ABSTRACT FROM AUTHOR]