Abstract:[Background] Wild Grammoplites scaber is nutritious, but the microorganisms in its gastrointestinal tract (GI) are rarely studied. [Objective] To study the microbial community in GI of G. scaber, reveal the potential probiotics and pathogens, and thus to provide a reference for the regulation of beneficial microbes. [Methods] We collected the GI samples of G. scaber from the Pearl River Estuary and investigated them with the culture-independent method and pure culture method. [Results] Through high-throughput sequencing of V3 regions of 16S rRNA genes, a total of 456 operational taxonomic units (OTUs) were identified. At the phylum level, Proteobacteria and Firmicutes dominated the microbes in the GI. At the genus level, Clostridium was prevalent in the samples, accounting for 57.11%. The 16S rRNA gene-based phenotypic and functional prediction suggested that the GI of G. scaber harbored both probiotics and pathogens, and they tended to restrict each other. A total of 99 strains were screened out with 12 different selective media, and they belong to 13 genera, 10 families, 10 orders, 4 classes, and 3 phyla (Proteobacteria, Firmicutes, and Actinobacteria). The dominant group was Proteobacteria (50.51%), and Psychrobacter was the predominant genus. [Conclusion] This study reveals the microbial composition and diversity in the GI of wild G. scaber, laying a basis for the research on the core intestinal microflora of teleost. In addition, the probiotics and pathogens identified in this study can serve as a reference for the safety of food made with G. scaber and for the development of marine fishery.