Rahnella aquatilis as an emerging pathogen can cause bacterial enteritis in cyprinid fish such as crucian carp Carassius auratus. Currently, the characterisation of immune function and gut microbiome composition in C. auratus orally challenged by R. aquatilis were yet unknown. In this study, we therefore investigated the changes of histopathology, white blood cells (i.e., LEU, NEU and LYM), serum biochemical indicators (e.g., CRE, CK and CHO) and digestive enzyme activity (e.g., LYS, AST, ALT, GSH-Px and AKP), as well as complements and immune-related genes (e.g., C3, F2, LysC, TLR3, MyD88, TGF-β, TNF-α and IL-15) that were significantly altered after the oral administration of R. aquatilis KCL-5. Moreover, the gut microbiome composition and diversity were analysed by using 16S rRNA gene high-throughput sequencing analysis. The correlation analysis showed that the high abundance of phylum Proteobacteria, Actinobacteria, Firmicutes and Fusobacteria was related to the pathogenesis of enteritis caused by oral infection. KEGG enrichment analysis indicated that fatty acid, carbon and pyruvate metabolism were significantly increased pathways (p <
0.05). To our best knowledge, this is a rare report of physicochemical properties and gut microbiome in C. auratus by R. aquatilis infection, which will provide a scientific reference for the clinical diagnosis and prevention of bacterial enteritis in cyprinid fish.