The mud crab Scylla paramamosain is introduced to inland saline-alk. water, but its survival is still a challenge.In this study, we performed a NMR-based metabolomic anal. of the gut of the mud crab during the saline-alk. water pond culture and dietary supplementation with trimethylamine N-oxide (TMAO), which was depleted in the gut, using a simulated saline-alk. culture of the mud crab.We observed that the gut metabolite profile of mud crabs was significantly affected by saline-alk. stress.Following a multivariate data anal., we identified significantly changed metabolites associated with saline-alk. stress such as the depleted succinate, trimethylamine, and TMAO as well as the accumulated taurine, putrescine, and glucose, indicating a disruption in osmoregulation and energy metabolismImportantly, dietary supplementation of 0.2 % TMAO promoted the survival of the mud crab in saline-alk. water, and improved the activities of Na+/K+-ATPase, vacuolar-type ATPase, and carbonic anhydrase in the gill as well as transcriptional expressions of pk, hk, fbp, and pepck in the hepatopancreas, which suggests the vital role of TMAO in osmoregulation, acid-base homeostasis, and glucose metabolismThese findings provide a potential alternative of feed additive for the mud crab cultured in saline-alk. water.