The effects of replacing fish meal (FM) with low-gossypol cottonseed meal (LCSM) on the intestinal barrier in different segments (PI, proximal intestine; MI, middle intestine; DI, distal intestine) were studied in juvenile golden pompano (Trachinotus ovatus) using 6-week feeding trials. FM of 0% (diet_1), 20% (diet_2), 40% (diet_3) and 60% (diet_4) was replaced by LCSM. The FM-based diet (diet_1) containing 25% FM was used as control group. The intestinal morphology, mRNA expression of tight junctionrelated genes (ZO-1, Occludin, Claudin-12), antioxidant enzyme-related genes (CAT, GPx, GR, CuZnSOD) and non-specific immune response-related genes (IL-8, TNFα, C4) and microbiota of different intestine segments were detected. The results showed that the number and length of microvilli and intestinal wall thickness decreased significantly in PI of diet_3 and diet_4 groups. Meanwhile, mRNA expression of Occludin, Clauding-12 and C4 was up-regulated in PI and DI of diet_3 group; the expression of GR and CuZnSOD was up-regulated in PI and MI with increasing LCSM replacement; the expression of TNFα was up-regulated in the three segments of diet_3 group. Further, 16S rRNA amplicon sequencing revealed a significant reduction in the richness and diversity of intestinal microbial in diet_3 and diet_4 groups. At the family level and genera level, the abundance of Nocardiaceae and Rhodococcus was up-regulated in the three segments of diet_3 and diet_4 groups respectively. These results suggested that the replacement of LCSM by more than 20% causes the intestinal barrier function of juvenile golden pompano to be affected. These provide a reference for sustainable aquaculture.