Aging is a major cause of bone loss and osteoporosis. Diallyl trisulfide (DATS), one of the main organic sulfides in garlic oil, has been shown to alleviate arthritis in mice. However, further research is still needed to determine how DATS affects bone formation and bone loss in aging mice. Here, we established a mouse model of natural aging for dietary DATS intervention. DATS treatment improved the bone microstructure, including the disorganized arrangement of bone trabeculae and promoted collagen synthesis, as confirmed by micro-CT and histological analyses. The abundance of beneficial bacteria for bone formation, such as Clostridiaceae and Erysipelotrichaceae, and the microbial diversity and community richness were all altered by DATS, according to 16S rRNA sequencing data. 14 potential biomarkers and 9 important metabolic pathways were examined using serum metabolomics analysis. Additionally, there has been a significant reduction in sphingosine, which is directly associated with bone metabolism. The level of sphingosine and relative abundance of Clostridium were found to be negatively correlated by correlation analysis, indicating that bacteria may regulate bone reconstruction via influencing metabolites. Furthermore, Runx2 and β-catenin gene expression levels increased in bones, which may be related to the ameliorative mechanism of DATS. Our results suggested that DATS may prevent age-related bone loss by upregulating osteogenic gene expression through altering gut microbes and serum metabolism.