Nile tilapia (Oreochromis niloticus) is a well-known economic fish species and can grow well under optimum environmental conditions. However, stressful rearing conditions are expected to deteriorate the performance and health condition of fish. Hence, the present study investigated the physiological and ion changes of Nile tilapia under salinity stress. Fish of 200 ± 20 g were divided into three groups where the control group reared in water with 0 ppt salinity, while the second and third groups were raised in water with low salinity (10 ppt) and high salinity (15 ppt), respectively. Blood dissolved oxygen and gases (pH, PO2, PCO2, TCO2, and HCO3−) significantly differed in different groups after 5 and 10 days (P < 0.05). The salinity stress affected the Hb, PCV, Na+, K+, Ca++, and lactate significantly (P < 0.05). Serum cortisol, triiodothyronine, and free triiodothyronine concentrations after 5 and 10 days were significantly affected (P < 0.05) in salinity stressed groups compared to the control group. Serum malondialdehyde showed a significant increase (P < 0.05) after 5 days in the stressed groups, then became non-significant after 10 days compared to the control group. The relative gene expression of Na+-K+-ATPase and cytosolic carbonic anhydrase were significantly upregulated in salinity stressed groups (P < 0.05). The histopathological images of Nile tilapia exposed to high salinity stress revealed damage in gills, liver, and kidney tissues, which confirmed the harmful effects of increased salinity. Thus, the obtained results demonstrated that the adverse effects of high salinity stress on the health status of Nile tilapia.