Abstract: The grass carp (Ctenopharyngodon idella) is one of the major freshwater aquaculture species in China, contributing significantly to the aquaculture sector with a national production nearing 6 million tons in 2023. However, viral hemorrhagic disease caused by grass carp reovirus (GCRV) poses a significant disease affecting grass carp farming, often resulting to substantial economic losses in the industry. Macroalgaes, characterized by their diversity, wide oceanic distribution, and high productivity, harbor a wealth of natural bioactive compounds including algal polysaccharides, alginates, algal polyphenols, and dietary fiber. These substances can regulate the nutrients metabolism and absorption in animals and exhibit proven antiviral and antioxidant activities. Currently, macroalgae are used as natural feed components in aquaculture to bolster fish disease resistance. However, the potential impact of incorporating macroalgae on GCRV infection in grass carp via the modulation of gut microbiota and host immunity remains uncertain. In this study, a dried powder comprising Sargassum hemiphyllum, Asparagopsis taxiformis, and Gracilaria lemaneiformis was added to grass carp feed for two months, followed by intraperitoneal GCRV infection. High-throughput 16S rRNA gene sequencing was used to analyze the gut microbiota, with subsequent quality control and assembly of raw sequencing reads to obtain high-quality paired-end sequences. Chimerism sequences was identified and removed, while sequences exhibiting a similarity above a defined threshold of 97 were clustered into Operational Taxonomic Units (OTUs). These OTUs were annotated by using RDP classifier based on Silva 16S rRNA database. Subsequently, species annotation was conducted, resulting in the acquisition of raw abundance data. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of complement component 5 (C5), C5a, and C5a receptor (C5aR) in the serum to assess the host immune response. The results showed that macroalgae supplementation significantly increased the Shannon and Simpson indices of gut microbiota in grass carp. NMDS, Venn diagram, and heatmap analysis indicated a significant restructuring of the gut microbiota following macroalgae supplementation. This restructuring led to an increased presence of distinct bacterial species primarily associated with dietary fiber fermentation and carbohydrate digestion. Furthermore, it contributed to a delayed decline in gut microbiota diversity observed on the initial day post-GCRV infection. Differential analysis, based on Bray-Curtis distance, was performed for the gut microbiota before GCRV infection (day 0, G0) and on days 4 and 7 post-infection (G4 and G7). The results revealed a significant alteration in the gut microbiota structure of the control group between days 4 and 7 post-GCRV infection, while no significant changes were observed in the macroalgae supplementation group. Specifically, the relative abundance of Vibrio in the intestinal tract reduced significantly in the groups fed with S. hemiphyllum and G. lemaneiformis. In the S. hemiphyllum group, the relative abundance of Cetobacterium decreased, while that of Bacteroides increased. Co-occurrence network analysis of the gut microbiota from the infected grass carp indicated that macroalgae enhanced the network complexity of the microbiota, alleviating the impact of GCRV on their gut microbiota. Analysis of complement expression in the serum of infected grass carp revealed that S. hemiphyllum upregulated C5a expression on day 1 post-infection, while G. lemaneiformis downregulated C5a expression. Spearman correlation analysis suggested that after GCRV infection, macroalgae might influence the expression of C5, C5a, and its receptor (C5aR) by regulating beneficial and pathogenic bacteria in the intestine. In conclusion, the addition of macroalgae alleviates the impact of GCRV on the gut microbiota of grass carp, with G. lemaneiformis contributing to the reduction of inflammatory responses. These findings could pave the way for novel strategies in the prevention and management of hemorrhagic disease in grass carp, offering significant practical implications for the aquaculture industry.