The allicin diallyldisulfid- S-oxide, a major garlic organosulfur compound (OSC) in crushed garlic ( Allium sativum L.), possesses antibacterial effects, and influences gut bacteria. In this study, we made allicin-free garlic (AFG) extract and investigated its effects on gut microbiome. C57BL/6N male mice were randomly divided into 6 groups and fed normal diet (ND) and high-fat diet (HFD) supplemented with or without AFG in concentrations of 1% and 5% for 11 weeks. The genomic DNAs of feces were used to identify the gut microbiome by sequencing 16S rRNA genes. The results revealed that the ratio of p- Firmicutes to p- Bacteroidetes increased by aging and HFD was reduced by AFG. In particular, the f- Lachnospiraceae, g- Akkermansia, and g- Lactobacillus decreased by aging and HFD was enhanced by AFG. The g- Dorea increased by aging and HFD decreased by AFG. In addition, the ratio of glutamic-pyruvic transaminase to glutamic-oxaloacetic transaminase (GPT/GOT) in serum was significantly increased in the HFD group and decreased by AFG. In summary, our data demonstrated that dietary intervention with AFG is a potential way to balance the gut microbiome disturbed by a high-fat diet.