In this study, we investigated the effects of dietary myo-inositol on the growth and intestinal physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp (221.83 ± 0.84 g) were fed six diets containing graded levels of myo-inositol (27.0, 137.9, 286.8, 438.6, 587.7 and 737.3 mg/kg) for 10 weeks. After the growth trial, fish were challenged with Aeromonas hydrophila for 14 days. The results indicated that compared with optimal myo-inositol levels, myo-inositol deficiency (27.0 mg/kg diet): (1) decreased glutathione (GSH) contents and antioxidant enzymes activities, and down-regulated the mRNA levels of antioxidant enzymes [not glutathione-S-transferase (gst) p1 and gstp2] and NF-E2-related factor 2 (nrf2), whereas up-regulated the reactive oxygen species (ROS), malondialdehyde (MDA) and protein carbonyl (PC) contents, and the mRNA levels of Kelch-like-ECH-associated protein 1 (keap1) in three intestinal segments of young grass carp (P < 0.05). (2) Up-regulated cysteinyl aspartic acid-protease (caspase)-2, -3, -7, -8, -9, apoptotic protease activating factor-1 (apaf-1), Bcl2-associated X protein (bax), fas ligand (fasl), gen-activated protein kinase (p38mapk) and c-Jun N-terminal protein kinase (jnk) mRNA levels, whereas down-regulated B-cell lymphoma-2 (bcl-2), inhibitor of apoptosis proteins (iap) and myeloid cell leukemia-1 (mcl-1) mRNA levels in three intestinal segments of young grass carp (P < 0.05). (3) Down-regulated mRNA levels of cell cycle proteins cyclin b, cyclin d, cyclin e and E2F transcription factor 4 (e2f4) in three intestinal segments of young grass carp (P < 0.05). (4) Down-regulated the mRNA levels of zonula occludens (zo) 1, zo-2, occludin, claudin-b, -c, -f, -3c, -7a, -7b as well as -11, and up-regulated the mRNA levels of claudin-12, -15a (not -15b) and myosin light chain kinase (mlck) in three intestinal segments of young grass carp (P < 0.05). All above data indicated that dietary myo-inositol deficiency could damage physical barrier function in three intestinal segments of fish. Finally, the myo-inositol requirements based on the percent weight gain (PWG), reactive oxygen species (ROS) contents in the proximal intestine (PI), relative mRNA levels of caspase-2 (PI), cyclin b (MI) as well as claudin-b (PI) were estimated to be 276.7, 304.1, 327.9, 416.7 and 313.2 mg/kg diet, respectively.