Objective To study the hematopoietic stem cell injury(HSC) induced by busulfan. Methods C57BL/6 mice were treated with i. p. injection of 20 mg/ kg or 40 mg/ kg busulfan. All mice were euthanized at 15 days and 30 days after busulfan treatment for analysis of the peripheral blood cell counts, bone marrow cellularity and HPC (LKS-, lineage-sca1^- c-kit⁺ ), HSC (LSK, lineage-sca1⁺ c-kit⁺ ) and long-term HSC (CD34^- LSK, CD34^- lineage-sca1 ⁺ c-kit⁺ ) frequency. The colony-forming unit-granulocyte and macrophage (CFU-GM) ability of HPC was measured by colony-forming cell (CFC) assay, and the HSC self-renewal capacity was analyzed by single-cell colony-forming assay. Results The busulfan administration decreased the WBC, RBC and PLT compared with control mice. The HPC function (CFU-GM) was impaired (P < 0.05), and the HSC colony forming ability was decreased at 15 days after busulfan treatment (P < 0.05), whereas the body weight of the mice didn’ t change significantly after busulfan treatment. Conclusions Our findings suggest that busulfan can induce hematopoietic stem cell injury, and provide a support for the study of hematopoietic stem cell injury mechanism.