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The aim of this study was to determine the impact of replacing fishmeal (FM) with soybean meal (SBM) in the diet of orange-spotted grouper on growth and intestinal tissue structure. FM was replaced with 0%, 50%, or 100% SBM (FM100, FM50, FM0, respectively) to create three iso-nitrogenous and iso-lipidic experimental diets. Five orange-spotted grouper (initial mean body weight: 59 ± 3 g) were stocked into 150-L tanks on a recirculating system, and each dietary treatment level was assigned to three replicate tanks. Fish were fed twice daily for 30 days. Weight gain rate, feed intake and feed efficiency significantly decreased as the proportion of SBM increased. Villus height decreased while lesioning, damage and mixed leucocytes infiltration significantly increased in the intestines of fish fed the FM0 and FM50 diets (P < 0.05). With increasing SBM, the expression levels of IL-1β and IL-16 mRNA significantly increased, while the expression level of IL-10 mRNA significantly decreased (P < 0.05). In conclusion, increasing the amount of soybean meal in the diets of orange-spotted grouper leads to decrease growth performance and increase intestinal harm.