An 8-week feeding trial was conducted to investigate the effects ofC. butyricumon the growth performance, microbiota, immunity response, and disease resistance in hybrid grouper fed with cottonseed protein concentrate (CPC) replacement of fishmeal. Six groups of isonitrogenous and isolipid diets were formulated including a positive control group (50% fishmeal, PC), a negative control group (CPC replaced 50% of fishmeal protein, NC), andClostridium butyricumsupplemented with 0.05% (C1, 5 × 108CFU/kg), 0.2% (C2, 2 × 109CFU/kg), 0.8% (C3, 8 × 109CFU/kg), and 3.2% (C4, 3.2 × 1010CFU/kg), respectively, to the NC group. The results showed that weight gain rate and specific growth rate were significantly higher in the C4 group than that in the NC group (P< 0.05). After supplementation withC. butyricum, the amylase, lipase, and trypsin activities were significantly higher than the NC group (P< 0.05; except group C1), and the same results were obtained for intestinal morphometry. The intestinal proinflammatory factors were significantly downregulated, and the anti-inflammatory factors were significantly upregulated in the C3 and C4 groups compared with the NC group after supplementation with 0.8%-3.2%C. butyricum(P< 0.05). At the phylum level, the PC, NC, and C4 groups were dominated by the Firmicutes and the Proteobacteria. At the genus level, the relative abundance ofBacillusin the NC group was lower than that in the PC and C4 groups. After supplementation withC. butyricum, grouper in the C4 group showed significantly higher resistance toV. harveyithan the NC group (P< 0.05). Above all, taking into account the effects of immunity and disease resistance, it was recommended to supplement 3.2%C. butyricumin the diet of grouper fed the replacement of 50% fishmeal protein by CPC.