The largemouth bass is an economically important fish species in our country, which exhibits a certain degree of salt tolerance, rapid growth, adaptability, and superior meat quality. It has great potential for aquaculture in saline-alkaline water. Therefore, in light of the greater expansion of aquaculture to saline-alkaline regions, it is of vital importance to explore the osmoregulation and physiological responses of largemouth bass under different salinity stresses. Although previous studies have highlighted its moderate salinity tolerance, the physiological and molecular responses to graded salinity challenges remain unclear, particularly regarding ion regulation, oxidative stress, and tissue-specific gene expression. This study systematically investigated the effects of salinity stress (0, 5, 10, and 15) on osmoregulation, antioxidant capacity, histology, and NKCC1a expression in largemouth bass, with the aim of establishing a comprehensive framework for evaluating its adaptability to saline conditions and developing sustainable aquaculture strategies. Largemouth bass, with an average weight of (20.3±1.3) g, was subjected to stress experiments under various salinity conditions (0, 5, 10, and 15). The initial salinity of each group was 0, followed by an increase of 2 every 12 h. After reaching the specified concentrations for 24 h, three fish were collected from each of the three experimental groups. Serum biochemical indicators, osmoregulatory enzyme activities, antioxidant enzyme activities, pathological tissue changes, and the relative expression levels of NKCC1a were assessed. Statistical evaluations included one-way ANOVA and Duncan’s multiple comparison test (significance at P＜0.05). Our results showed that the serum osmolality, Na⁺ concentration, and Cl^– concentration of largemouth bass increased to varying degrees with the rise in salinity, with significant differences between the salinity 15 group versus the 10, 5, and control groups (P＜0.05). Correlation analysis showed that osmolality has a strong positive correlation with Na+ and Cl⁻ (r=0.88 and r=0.96), which reflects the strategy of osmoregulation in fish by actively absorbing Na+ and Cl^– to cope with the external high-salinity environment. Serum cortisol concentrations increased with higher salinity, indicating that cortisol actively participates in osmoregulation. The cortisol concentration at salinity 10 was significantly higher than that in the other three groups (P＜0.05), indicating that high-salinity stress promotes the release of cortisol. Correlation analysis found a strong positive correlation between cortisol (COR) and Na⁺ , Cl^– , and Na⁺ /K⁺ -ATPase (NKA) (r＞0.9), further supporting its key role in coping with salinity stress. Notably, NKA and Ca²⁺/Mg²⁺-ATPase (CMA) activities peaked at salinity 10, but declined sharply at salinity 15, suggesting enzymatic dysfunction under extreme salinity. Superoxide dismutase (SOD) activity increased progressively with salinity and peaked at salinity 15 (210.57 U/mg), whereas catalase (CAT) activity peaked at salinity 10 (35.72 U/mg) before declining, indicating oxidative stress overload at higher salinities. In this study, chloride cells in the gills gradually increased and enlarged as salinity increased, while the gill filaments were gradually damaged, accompanied by shedding. Intestinal tissue showed an increase in goblet cells with rising salinity, in addition to damage and shedding of intestinal villi occurred. This indicates that the largemouth bass responds to osmotic stress by enhancing ion transport (gills) and mucin barriers (intestines). However, the pathological features of gill filament shedding and intestinal villus breakage at salinity 15 suggest that tissue repair capacity may be inhibited by high salinity. Notably, the hyperplasia of intestinal goblet cells observed in this study may have dual implications. On the one hand, it alleviates osmotic shock through mucin secretion, on the other hand, it may interfere with nutrient absorption efficiency, providing histological evidence for subsequent research on the decline in growth performance under salinity stress. Moreover, the expression levels of NKCC1a in the gills and intestines were tissue-specific, and the expression levels of NKCC1a at salinity 5, 10, and 12 were consistently significantly higher than those in the control group (P＜0.05). This study systematically analyzed the physiological and molecular adaptation mechanisms of the largemouth bass in response to different salinity stress. These results demonstrate that largemouth bass effectively modulate ion regulatory and antioxidant systems at salinity 5 and 10, but suffer from significant physiological impairment at salinity 15. The tissue-specific upregulation of NKCC1a and its correlation with cortisol levels suggest a coordinated molecular response to salinity. This study provides critical insights into the salinity threshold of this species (10), identifying cortisol and NKCC1a as potential biomarkers for stress assessment. The results of this study provide a reference and support data for the culture and development of largemouth bass in brackish water environments.