Simple SummaryThis study analyzed the dynamic changes in the intestinal microbiome of pigs after being infected with the highly virulent African swine fever virus (ASFV) genotype II strain. The fecal microbiomes of infected pigs were thoroughly investigated according to the four phases of ASFV infection: before infection, primary phase, clinical phase, and terminal phase. As a result, the richness indices (ACE and Chao1) significantly decreased in the terminal phase. The relative abundances of short-chain-fatty-acids-producing bacteria, such asRuminococcaceae,Roseburia, andBlautia, significantly decreased during ASFV infection. On the other hand, the abundance ofProteobacteriaandSpirochaetesincreased. The predicted functional analysis using PICRUSt revealed a significantly reduced abundance of 15 immune-related pathways in the ASFV-infected pigs. This study provides evidence for further understanding the ASFV–pig interaction and suggests that changes in gut microbiome composition during ASFV infection may be associated with the status of immune modulation.AbstractThe factors that influence the pathogenicity of African swine fever (ASF) are still poorly understood, and the host’s immune response has been indicated as crucial. Although an increasing number of studies have shown that gut microbiota can control the progression of diseases caused by viral infections, it has not been characterized how the ASF virus (ASFV) changes a pig’s gut microbiome. This study analyzed the dynamic changes in the intestinal microbiome of pigs experimentally infected with the high-virulence ASFV genotype II strain (N = 4) or mock strain (N = 3). Daily fecal samples were collected from the pigs and distributed into the four phases (before infection, primary phase, clinical phase, and terminal phase) of ASF based on the individual clinical features of the pigs. The total DNA was extracted and the V4 region of the 16 s rRNA gene was amplified and sequenced on the Illumina platform. Richness indices (ACE and Chao1) were significantly decreased in the terminal phase of ASF infection. The relative abundances of short-chain-fatty-acids-producing bacteria, such asRuminococcaceae,Roseburia, andBlautia, were decreased during ASFV infection. On the other hand, the abundance ofProteobacteriaandSpirochaetesincreased. Furthermore, predicted functional analysis using PICRUSt resulted in a significantly reduced abundance of 15 immune-related pathways in the ASFV-infected pigs. This study provides evidence for further understanding the ASFV–pig interaction and suggests that changes in gut microbiome composition during ASFV infection may be associated with the status of immunosuppression.