Glycinin, a major anti‐nutrient in soybean meal, may trigger enteritis and oxidative stress in fish with overconsumption. However, its impact on intestinal inflammation and underlying signaling mechanisms in Larimichthys crocea remains unclear. The purpose of this study was to assess the effects of glycinin on growth, antioxidant function, inflammatory response, and gut microbiota. Four isonitrogenous and isolipidic experimental feeds were prepared, supplemented with 0%(G0), 3%(G3), 6%(G6), and 12%(G12) glycinin, respectively, and fed to juvenile L. crocea for 10 weeks. The findings indicated that dietary glycinin markedly decreased the growth, feed utilization, and survival rate of L. crocea. In addition, L. crocea fed with G6 and G12 diets showed lower trypsin activity compared with those fed with G0 and G3 diets. Similarly, fish fed with G6 and G12 diets showed higher malondialdehyde content and lower superoxide dismutase activity in the liver compared with those fed with G0 and G3 diets, suggesting that glycinin induced oxidative stress and led to the imbalance of the antioxidant system. Dietary glycinin significantly increased the mRNA expression levels of toll‐like receptors in the gut. Moreover, dietary glycinin resulted in a heightened expression of both C‐rel and p65 proteins, while also elevating the phosphorylation levels of JNK and ERK proteins, indicating that glycinin activated the MAPK/NF‐κB signaling route. In addition, dietary glycinin increased the abundance of pathogenic bacteria in the gut, including Bacteroides and Streptococcus, and reduced the Bifidobacterium abundance. The findings suggested that L. crocea is highly sensitive to glycinin, and a 3% level can cause growth decline and enteritis.