A 3‐hr experiment was conducted to investigate the effects of dietary myo‐inositol (MI) supplementation on survival, immune response and antioxidant abilities in Litopenaeus vannamei under acute hypoxia stress. Six practical diets were formulated with supplementation of graded levels (control group 0, 0.1, 0.2, 0.4, 0.8 and 1.6 g/kg dry diet) of MI and were randomly assigned to triplicate groups of L. vannamei (mean weight 0.40 ± 0.00 g) for 8 weeks. Ten healthy shrimp (final mean weight approximately 11–14 g) randomly selected from each tank were exposed to hypoxia stress after feeding trial. After 3‐hr acute hypoxia stress, survival of shrimp fed MI‐supplemented diets (except 0.1 and 0.4 g/kg diets) was significantly increased compared with the control group. Shrimp fed control diet had lower activities of alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (T‐AOC) and glutathione peroxidase (GPX), and higher malondialdehyde (MDA) and protein carbonyl (PC) contents in hepatopancreas than those fed the MI‐supplemented diets. In addition, mRNA expression levels of heat shock protein 70 (Hsp70), catalase (CAT) and penaeidin were significantly differentially regulated in hepatopancreas. In summary, dietary MI supplementation may have a positive effect on improving resistance to acute hypoxia stress of L. vannamei.