This study aimed to investigate the effects of tea polyphenol (TP) supplementation on attenuating Aeromonas hydrophila - induced intestinal barrier function injury in fish. A total of 540 healthy grass carp (187.49 ± 0.66 g) were randomly divided into six groups with three replicates and fed TP-supplemented diets at 0, 40, 80, 120, 160 and 200 mg/kg for 60 days. After the growth trial, 24 fish per group were randomly selected for a challenge test with A. hydrophila for 6 days. The results indicated that an appropriate level of TP (120 mg/kg diet) repair damaged the intestinal morphology of fish induced by A. hydrophila. Additionally, appropriate level of TP ameliorated the intestinal apical junctional complex partly, which was partly related to the up-regulation of the expressions of tight junction and adherent junction proteins via the RhoA/ROCK pathway in the fish intestine. Based on the results of this research, dietary TP supplementation possesses a protective function of the intestinal physical barrier in fish against A. hydrophila, making TP a functional feed additive for aquaculture and providing a solution for the issue of hazards to human health caused by antibiotic residues in aquatic food.