Berberine (BBR) is known for its strong antioxidant, anti-inflammatory, and capacity to preserve intestinal microbiota balance in fish. This study aimed to investigate the protective effects of berberine against copper-induced toxicity in the intestine of freshwater grouper Acrossocheilus fasciatus. The experiment involved four groups: a control group, a Cu group exposed to 0.02 mg/L Cu(2+), and two BBR groups fed with 100 or 400 mg/kg of berberine diets and exposed to the same Cu(2+) concentration. Three replicates of healthy fish (initial weight 1.56 +/- 0.10 g) were subjected to their respective treatments for 30 days. Results showed that none of the treatments significantly affected the survival rate, final weight, weight gain, and feed intake (P > 0.05). However, supplementation with 100 and 400 mg/kg of BBR significantly lowered the antioxidant activities, and glutathione peroxidase (gpx) and superoxide dismutase (sod) expression levels, as well as reduced malondialdehyde (MDA) content caused by Cu(2+) exposure (P < 0.05). Berberine inclusion significantly downregulated proinflammatory factors NLR family pyrin domain containing 3 (nlrp3), interleukin 1 beta (il1beta), interleukin 6 cytokine family signal transducer (il6st) but upregulated transforming growth factor beta 1 (tgfbeta1) and heat shock 70 kDa protein (hsp70) expression. Moreover, berberine at both levels maintained the intestinal structural integrity and significantly improved gap junction gamma-1 (gjc1) mRNA level compared to the Cu group (P < 0.05). Based on 16S rDNA sequencing, the richness and diversity of intestinal microbiota in different groups were not significantly influenced. Berberine reduced the Firmicutes/Bacteroidota ratio and stifled the growth of some specific pathogenic bacteria such as Pseudomonas, Citrobacter, and Acinetobacter, while boosting the richness of potential probiotic bacteria, including Roseomonas and Reyranella compared with the Cu group. In conclusion, berberine showed significant protective effects against Cu(2+)-induced intestinal oxidative stress, inflammation response, and microbiota disturbance in freshwater grouper. CI - Copyright (c) 2023 Elsevier Ltd. All rights reserved.