BACKGROUND: Osteoporosis, a debilitating condition characterized by decreased bone mass and increased fracture risk, requires novel insights into its molecular mechanisms for improved therapeutic approaches. In this study, we comprehensively explore the causal links between gut microbiota, immune cell regulation, and osteoporosis by integrating Mendelian randomization (MR), single-cell RNA sequencing (scRNA-seq), and bioinformatics analyses. METHODS: We employed a two-sample MR approach to investigate the causal associations between 412 gut microbiota species and two osteoporosis traits using data from the UK Biobank and Finnish cohorts. Additionally, 731 immune cell types were analyzed as potential mediators between the gut microbiota and osteoporosis. Bioinformatics analysis, including gene ontology (GO) and KEGG pathway enrichment, was used to assess the functional implications of differentially expressed genes. ScRNA-seq from publicly available datasets was conducted to profile the expression of key genes, including USP6NL, SELENOT, and TAF1A, in osteoporotic and control samples. RESULTS: The MR analysis identified significant causal relationships between the gut microbiota (notably the glyoxylate cycle) and osteoporosis outcomes. Furthermore, HLA-DR expression on hematopoietic stem cells (HSCs) was identified as a crucial immune cell mediator between the gut microbiota and osteoporosis, highlighting the immune-microbiota-bone axis. Differential expression analysis from scRNA-seq confirmed the upregulation of USP6NL, SELENOT, and TAF1A in osteoporotic samples. Functional enrichment analysis revealed that these genes play significant roles in pathways related to oxidative stress, calcium homeostasis, and immune modulation. These findings were validated through GTEX data integration, identifying USP6NL, SELENOT, and TAF1A as potential therapeutic targets for osteoporosis. CONCLUSIONS: This study provides novel insights into the interplay between gut microbiota, immune regulation, and bone metabolism in osteoporosis. The integration of Mendelian randomization, single-cell RNA sequencing, and bioinformatics analyses uncovers USP6NL, SELENOT, and TAF1A as key mediators and potential therapeutic targets in osteoporosis. These findings open up new avenues for personalized treatment strategies targeting the gut-immune-bone axis in osteoporosis management.