The effect of acute ammonia challenge on survival, immune response and antioxidant status of Litopenaeus vannamei pretreated with diets containing different inositol levels was investigated. Shrimp (initial mean weight 0.40 ± 0.00 g) were randomly allocated in 18 tanks (30 shrimp per tank) and triplicate tanks were fed with a control diet without myo-inositol (MI) supplementation (242.6 mg inositol kg-1 diet) or diets containing diverse levels of inositol (368.8, 459.7, 673.1, 993.8 and 1674.4  mg kg-1 diet) as treatment groups for 8-week. Randomly selected 10 shrimp per tank (final mean weight approximately 11.1-13.8g) were exposed to ammonia stress (total ammonia-nitrogen, 60.21  mg L-1) for 24 h after feeding trial. The results showed that after exposed to ammonia stress, survival rates of MI-supplemented groups were enhanced by 31-77% when compared with the control group. MI supplementation increased activities of alkaline phosphatase (AKP) and acid phosphatase (ACP) in plasma, and reduced its activities in hepatopancreas. It also enhanced activities of total antioxidant capacity (T-AOC), glutathione S-transferase (GST) and glutathione peroxidase (GPX) and content of reduced glutathione (GSH), and lowered malondialdehyde (MDA) and protein carbonyl (PC) content in plasma or hepatopancreas. In addition, mRNA expression levels of ferritin (FT), arginine kinase (AK), thioredoxin (Trx), heat shock protein 70 (Hsp70), catalase (CAT) and peroxiredoxin (Prx) were significantly differentially regulated in hepatopancreas owing to MI supplementation. Therefore, it suggested that L. vannamei pretreated with higher dietary inositol content may have better ammonia stress tolerance and antioxidant status after ammonia stress, and the optimum levels ranged from 459.7 to 993.8 mg inositol kg-1 when total ammonia-nitrogen concentration was 60.21  mg L-1.