Objective The aim of this study is to establish a research protocol clarifying the characteristic changes in major functional activities and cellular processes involved in bone marrow during aging through RNA sequencing analysis, and to identify potential targets for aging prediction and intervention. Methods Bone marrow cells were extracted from the bilateral tibiae and femur of three C57BL/6J male mice aged 2 months, 10 months, and 18 months. After red blood cell lysis, RNA was extracted for RNA sequencing analysis. Results The results of gene expression and Venn analysis showed that the changes in gene expression from 2 months to 10 months were predominantly down-regulated, but mainly up-regulated from 10 months to 18 months. During the maturation and development of mice, gene expression undergone a change from mainly downregulation to mainly upregulation. According to the function and enrichment analysis of KEGG and GESA analysis, the bone marrow tissues of mice at different ages have significant expression differences in the "immune system", "development and regeneration", "transport and catabolism", "cell growth and death" and other pathways. Specifically, pathways related to inflammatory responses, cytoskeletal connections, and DNA damage repair showed sustained up-regulation, while hematopoietic function maintenance exhibited a continuous down-regulation trend. Additionally, immune regulation functions displayed fluctuating changes. Protein interaction networks constructed from differentially expressed genes, such as up-regulated genes Bmpr1a and Inhba, down-regulated genes Dntt and Ccnd1, and down-regulated genes Col1a1, Col1a2, Fcgr1, Fyn, Lgmn, Ctsl, Ctsk, Ctss, Gnail, Myl4, and Ccr5, demonstrated interconnected and interactive relationships. Conclusion 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 mouse aging. It identifies aging-related genes and signaling pathways, providing new strategies for delaying aging and preventing aging-related diseases.