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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 junction‐related genes (ZO‐1, Occludin, Claudin‐12), antioxidant enzyme‐related genes (CAT, GPx, GR, CuZnSOD) and non‐specific immune response‐related genes (IL‐8, TNF‐α, 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 TNF‐α 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. [ABSTRACT FROM AUTHOR]