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The aim of this experiment was to investigate the mechanism of the improved ammonia-N tolerance in Litopenaeus vannamei induced by dietary supplementation with freeze-dried powder of Ampithoe sp. (FDPA). In the present study, shrimp were divided into three groups and fed an FDPA-supplemented diet for 0 days (S0 group), 21 days (S21 group) or 42 days (S42 group). Then, the three groups of shrimp were exposed to ammonia-N (1.61 mg/L nonionic ammonia) for 96 h, and changes in oxidative stress, endoplasmic reticulum (ER) stress and lipid metabolism in the hepatopancreas were investigated. The cumulative mortality rates of the S0, S21 and S42 groups at the end of the ammonia-N stress test were 46.7 %, 30.0 % and 33.3 %, respectively. The activity levels of superoxide dismutase (SOD) and catalase (CAT) in the hepatopancreas of shrimp were increased in the FDPA diet groups compared with the S0 group. The level of malondialdehyde (MDA) and the mRNA expression of binding protein (Bip), eukaryotic initiation factor 2α (eIF2α), inositol requiring enzyme-1 (IRE1), spliced form of X box binding protein 1 (XBP1s) and activating transcription factor 4 (ATF4) were significantly decreased in the hepatopancreases of the S21 and S42 groups compared with those of the S0 group. Moreover, the activity of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and malonyl-CoA (MCoA) was decreased; the activity of carnitine palmitoyltransferase (CPT) was increased; and the levels of free fatty acid (FFA) and triglyceride (TG) were decreased in the hepatopancreases of the shrimp fed the FDPA diet compared with those of the shrimp fed the control diet. The results indicated that the FDPA diet could alleviate the oxidative stress and ER stress induced by ammonia-N exposure, ensure the normal metabolism and energy supply of lipids in the hepatopancreas, and thus enhance the ammonia-N tolerance of L. vannamei. Keywords: Ammonia-N tolerance, Litopenaeus vannamei, Oxidative stress, Endoplasmic reticulum stress, Lipid metabolism