A 56-day feeding trial was conducted to study the effects of cottonseed protein concentrate (CPC) as fishmeal (FM) replacement on growth, digestive proteinase, intestinal morphology and microflora in pearl gentian grouper (♀Epinephelus fuscoguttatus × ♂Epinephelus lanceolatu). FM of 0% (FM), 12.00% (R12), 24.00% (R24), 36.00% (R36), 48.00% (R48) and 60.00% (R60) was replaced with CPC, to prepare six diets and feed groupers. Weight gain (WG) and specific growth rate (SGR) were highest in the R24 group, and the lowest feed coefficient rate (FCR) was observed in this group compared with the other groups (p < .05). Pepsin activity of the R12 group and chymotrypsin activity of the R36 group reached significant maximum (p < .05). With increasing CPC replacement, the plica height and width and the lamina propria thickness of intestine decreased significantly (p < .05). 16S rDNA amplicon sequencing suggested dominant intestinal microflora of grouper subjected to different treatments was similar. At the phylum level, the abundance of Proteobacteria, Firmicutes, Verrucomicrobia, Bacteroidetes and Actinobacteria was high, while Photobacterium, Pseudoxanthomonas, Romboutsia, Akkermansia, Vibrio and Bifidobacterium were the dominant taxa at the genus level. The abundance of probiotics (Bifidobacterium) decreased, whereas the abundance of opportunistic pathogen (Proteobacteria, Actinobacteria and Vibrio) increased. These results suggested that CPC replacement affected growth, digestive proteinase activity, intestinal morphology and microflora structure in grouper. Under the conditions of this experiment, the fishmeal replacement with CPC (24% level) yielded the most satisfactory results, providing reference for sustainable aquaculture. K E Y W O R D S cottonseed protein concentrate, digestive proteinase, fishmeal replacement, growth, intestinal microflora, intestinal morphology