Simple SummaryThe mammalian gut microbiota is an indispensable part of host health. The gut microbiota plays a crucial role in nutrient digestibility, preventing colonization of pathogens and maintaining the host immune system. Host genetics has been conclusively shown to closely related to gut microbiota. Inbreeding can cause a decrease of the host’s genetic diversity, however, remarkably little is understood about the gut microbiota of pigs during inbreeding. The Banna minipig inbred is the world’s first successful large mammalian experimental animal inbred line since 1980 from full and half-siblings of the Diannan small-ear pig. Now, Banna minipig inbred has been inbred for over 37 generations, and the inbreeding coefficient is more than 99%. This study is the first to characterize and compare the composition and function of gut microbiota between the Diannan small-ear pig and Banna minipig inbred, aiming to better understand the influence of inbreeding on the gut microbiota.The gut microbiota coevolve with the host and can be stably transmitted to the offspring. Host genetics plays a crucial role in the composition and abundance of gut microbiota. Inbreeding can cause a decrease of the host’s genetic diversity and the heterozygosity. In this study, we used 16S rRNA gene sequencing to compare the differences of gut microbiota between the Diannan small-ear pig and Banna minipig inbred, aiming to understand the impact of inbreeding on the gut microbiota. Three dominant bacteria (Stenotrophlomonas, Streptococcus, and Lactobacillus) were steadily enriched in both the Diannan small-ear pig and Banna minipig inbred. After inbreeding, the gut microbiota alpha diversity and some potential probiotics (Bifidobacterium, Tricibacter, Ruminocaccae, Christensenellaceae, etc.) were significantly decreased, while the pathogenic Klebsiella bacteria was significantly increased. In addition, the predicted metagenomic analysis (PICRUSt2) indicated that several amino acid metabolisms (‘‘Valine, leucine, and isoleucine metabolism’’, ‘‘Phenylalanine, tyrosine, and tryptophan biosynthesis’’, ‘‘Histidine metabolism’’) were also markedly decreased after the inbreeding. Altogether our data reveal that host inbreeding altered the composition and the predicted function of the gut microbiome, which provides some data for the gut microbiota during inbreeding.