The aim of this study was to investigate the effect of graded levels of vitamin E (0-225 mg/kg) on gill immune response and physical barriers, and relative mRNA levels of signalling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin E supplementation, vitamin E deficiency (1) increased the rate of gill rot morbidity and aggravated gill histopathological injuries following F. columnare infection (P < 0.05); (2) decreased LA and ACP activities, complement component 3, complement component 4 and IgM contents and down-regulated antimicrobial peptides (liver-expressed antimicrobial peptide-2A, -2B, hepcidin, beta-defensin) and IgT mRNA levels (P < 0.05); (3) down-regulated the mRNA levels of anti-inflammatory cytokines and the signalling molecules inhibitory protein kappa B alpha, the target of rapamycin and ribosome protein S6 kinase 1 (P < 0.05) and up-regulated the mRNA levels of pro-inflammatory cytokines and the signalling molecules NF-kappa B p65, I kappa B kinase alpha (IKK alpha), IKK beta and eIF4E-binding protein 1 (P < 0.05); (4) decreased activities of anti-superoxide anion, Cu/ZnSOD, MnSOD, GPx, GST and GR, and down-regulated mRNA levels of those antioxidant enzymes and signalling molecule NF-E2-related factor 2 in the gills of fish (P < 0.05); (5) down-regulated the relative mRNA levels of B cell lymphoma-2 protein, and up-regulated the relative mRNA levels of caspase-2, -3, -7, -9, Bcl-2 associated X protein and apoptotic protease activating facter-1 (P < 0.05); (6) down-regulated the relative mRNA levels of tight junction proteins (occludin, zonula occludens-1, ZO-2, claudin-3, -b, -c and -11a) and up-regulated signalling molecule myosin light chain kinase mRNA levels (P < 0.05). In conclusion, vitamin E deficiency disrupted gill immune barriers and physical barrier function via impairing gill immunity and antioxidant capacity, inducing apoptosis and changing tight junction protein transcription abundances and the related signalling molecules in the gills of fish. The optimal vitamin E requirements for against gill rot morbidity of grass carp (266-1026 g) were estimated to be 139.56 mg/kg diet. Meanwhile, based on immune indicator (ACP activity) and antioxidant indicator (MDA content), the optimal vitamin E requirements for young grass carp were estimated to be 130.67 and 128.53 mg/kg diet, respectively.