<p>Global climate change has posed significant challenges to the aquaculture industry, leading to the emergence of novel diseases. A recently emerging disease, termed the overwintering syndrome, has been reported in freshwater fish aquaculture systems in China during the winter–spring transition. This syndrome is characterized by weight loss, decreased activity levels, and mass mortality. Currently, little is known about cause of the emerging disease. In this study, the gut microbiota of healthy and diseased channel catfish (<i>Ictalurus punctatus</i>) were analyzed using metagenomic sequencing. Diseased fish exhibited a notable reduction in bacterial diversity compared to healthy controls, while the viral richness was significantly higher. Additionally, random forest models based on gut microbiota composition revealed high accuracy in distinguishing between diseased and healthy fish. Several gut microbiota biomarkers with potential diagnostic value were identified, primarily consisting of rare taxa (relative abundance &lt; 5%), except for Alphaproteobacteira, Siphoviridae and Podoviridae. Furthermore, an increase in the prevalence and abundance of pathogens was observed in the intestines of diseased fish compared to healthy counterparts. Notably, <i>Vibrio cholerae</i> and <i>Dickeya dadantii</i> were enriched in the intestines of fish exhibiting the overwintering syndrome. Concurrently, genes involved in the synthesis of innate immune factors was markedly diminished in the gut microbiota of diseased fish, along with a reduction in the microbial hosts of these genes. Collectively, these findings elucidate the enrichment of pathogens and the attenuation of innate immunity in fish with the overwintering syndrome, correlating with alterations in the gut microbiota.</p>