Aquatic water with carbonate alkalinity presents a survival challenge to aquatic animals. As an economically important crab, large quantities of Eriocheir sinensis are cultured in carbonate-type saline-alkali ponds, while the toxic effect on E. sinensis from carbonate alkalinity is still unclear. In this study, untargeted liquid chromatography-mass spectrometry metabolomics was performed to investigate the metabolic change caused by culture alkalinity, and confirmed distinct physiological response under gradient alkalinities. There were 39 differential metabolites obtained in the low-alkalinity group (4.35 mmol/L) versus control group, and “arachidonic acid metabolism” was enriched as a core response pathway. 93 differential metabolites were identified in the high-alkalinity group (17.43 mmol/L) versus control group, and a complex response net was manifested through integrated analysis, building by “steroid hormone biosynthesis”, “phenylalanine, tyrosine and tryptophan biosynthesis”, “phosphonate and phosphinate metabolism”, “phenylalanine metabolism”, “mineral absorption”, “purine metabolism” and “carbon metabolism”. This indicated the mobilization of energy reserves and the suppression of protein and amino acid catabolism were manifested in E. sinensis gills to defense high alkalinity stress. In addition, the persistently regulation of key metabolites under various alkalinity, including diuretic compound “spironolactone” and the antiphlogistic compound “LXB4”, suggested anti-inflammatory action and excretion regulation were initiated to defend the stress.