Abstract Background Aquaculture plays an important role in global protein supplies and food security. The ban on antibiotics as feed additive proposes urgent need to develop alternatives. Gut microbiota plays important roles in the metabolism and immunity of fish, and has the potential to give rise to novel green inputs for fish culture. However, our understanding of fish gut microbiome is still lacking. Results We identified 575,856 non-redundant genes by metagenomic sequencing of the intestinal content samples of grass carp. Taxonomic and functional annotation of the gene catalogue revealed specificity of the gut microbiome of grass carp compared with mammals. Co-occurrence analysis indicated exclusive relations between the genera belonging to Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes, suggesting two independent ecological groups of the microbiota. The association pattern of Proteobacteria with the gene expression modules of fish gut and liver was consistently opposite to that of Fusobacteria, Firmicutes and Bacteroidetes, implying differential functionality of Proteobacteria and Fusobacteria/Firmicutes/Bacteroidetes. Therefore, the two ecological groups were divided into two functional groups, i.e., Functional Group 1: Proteobacteria; Functional Group 2: Fusobacteria/Firmicutes/Bacteroidetes. Further analysis revealed that the two functional groups differ in genetic capacity for carbohydrate utilization, virulence factors and antibiotic resistance. Finally, we proposed that the ratio of “Functional Group 2/Functional Group 1” can be used as a biomarker that efficiently reflects the structural and functional characteristics of the microbiota of grass carp. Conclusions The gene catalogue is an important resource for investigating the gut microbiome of grass carp. Multi-omics analysis provides insights into functional implications of the main phyla that comprise the fish microbiota, and shed lights on targets for microbiota regulation.