Objective To explore the damaging effect of busulfan on hematopoiesis and immune functions of KK/ upj-Ay / J (KKA^y) mice with type 2 diabetes mellitus. Methods Male KKA^y mice were randomly divided into busulfan treatment and vehicle control groups. The former was intraperitoneally injected with busulfan at a dose of 40 mg / kg, while the latter was injected with an identical volume of 5% dimethyl sulfoxide; B6 mice were grouped and treated in the same way. Fifteen days after administration, all mice were euthanized for peripheral blood count and classification of hematopoietic progenitor cell (HPC), hematopoietic stem cell (HSC), and long-term hematopoietic stem cell (LT-HSC) frequencies. The function of HPCs was assessed by colony-forming unit-granulocyte and macrophage ( CFU-GM) assay. Results Fifteen days after busulfan treatment, weight, white blood cell (WBC), red blood cell (RBC), blood platelet (PLT), lymphocyte (LYM), and neutrophil (NEU), as well as percentages of HPC, HSC, and LT-HSC in the bone marrow of KKA^y mice were significantly reduced. In addition, the number of CFU-GM was significantly reduced, although proportions of spleen and thymus cells were not significantly changed. After busulfan treatment, WBC, RBC, HGB, and LYM of KKA^y mice were significantly reduced compared with B6 mice, but proportions of PLT and HSC were significantly increased. PLT and HSC were significantly reduced in KKA^y mice compared with B6 mice, while the decrease of HPC in KKA^y mice was significantly enhanced compared with B6 mice. Conclusions Busulfan (40 mg / kg) impaired function of the hematopoietic system in mice. After busulfan was administered, WBC, RBC, HGB, and LYM of KKAy mice were severely damaged, PLT and HSC were less damaged and more tolerant, and the tolerance of HPC was reduced compared with B6 mice.