To explore the effect of carbonate alkalinity stress on Nile tilapia (Oreochromis niloticus), fish were cultured at 3 alkalinity levels (0, 2 and 3 g/L NaHCO3) for six weeks. Survival, weight gain, and specific growth rate were measured. Liver samples were collected for measuring superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content. Intestinal microbiota was analyzed using 16S rRNA gene sequencing, and metabolomic profiling identified differentially abundant metabolites. The results showed that the survival rate decreased in the fish exposed to 3 g/L NaHCO3, while weight gain and specific growth rate were not affected by the increased carbonate alkalinity. In the group exposed to 3 g/L NaHCO₃, activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and malondialdehyde (MDA) content in liver increased, while in 2 g/L NaHCO₃, only SOD and T-AOC activities rose compared to the control. Long-term carbonate alkalinity stress increased the abundance of Luteolibacter in intestine but decreased the abundance of Cetobacterium, Bacteroides, and Lactiplantibacillus. Metabolomics results showed that differentially abundant metabolites were highly enriched in pathways such as purine metabolism, vitamin B6 metabolism, lysine degradation, glycerophospholipid metabolism, and biosynthesis of cofactors in fish cultured at 3 g/L NaHCO3. These results suggested that carbonate alkalinity stress affects intestinal health by increasing the proportion of pathogenic bacteria and reducing beneficial bacteria. The correlation between N6,N6,N6-trimethyllysine, Ne,Ne-dimethyllysine, and Luteolibacter may represent an effective physiological adaptation and tissue repair pathway for Nile tilapia under carbonate alkalinity stress.