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.