Objective To establish a research protocol to clarify the characteristic changes in major functional activities and cellular processes involved in bone marrow during aging using RNA sequencing, and to identify potential targets for aging prediction and intervention. Methods Bone marrow cells were extracted from the bilateral tibiae and femurs of three C57BL/6J male mice aged 2, 10, and 18 months, respectively. After red blood cell lysis, RNA was extracted for sequencing analysis. Results The result of gene expression and Venn analysis showed that gene expression levels were predominantly down-regulated from 2 ~ 10 months, but mainly up-regulated from 10 ~ 18 months. Gene expression thus changed from mainly down-regulation to mainly up-regulation during maturation and development in mice. Kyoto encyclopedia of genes and genomes and gene set enrichment analyses indicated that bone marrow tissues in mice at different ages showed significant expression differences in the “immune system”“development and regeneration” “transport and catabolism” “cell growth and death” and other pathways. Specifically, inflammatory, cytoskeletal, and DNA repair pathways showed sustained activation, contrasting with progressive hematopoietic decline and fluctuating immune regulation. Enriched pathway screening revealed interactions among differentially expressed genes, such us upregulated genes Bmpr1a and Inhba, downregulated genes Dntt and Ccnd1, and downregulated genes Col1a1, Col1a2, Fcgr1, Fyn, Lgmn, Ctsl, Ctsk, Ctss, Gnail, Myl4, and Ccr5, involved in HSCs homeostasis, cell cycle, DNA repair, immune regulation, and apoptosis. Conclusions This study provides data on gene expression changes at the transcriptional level and offers a research strategy to explore the major characteristic changes in bone marrow during aging in mice. The result identify aging-related genes and signaling pathways, thus providing new strategies for delaying aging and preventing aging-related diseases.