Aim To investigate the effect of long-term high-fat diet (HFD) on the number of bone marrow-derived mesenchymal stem cell (BMSC) subsets in elderly mice and its mechanism. Methods Forty week old male C57BL/6 wild type (WT) mice were randomly divided into three groups:WT+normal diet (ND) group, WT+HFD group and WT+HFD+N-acetylcysteine (NAC) group. Forty week old male transgene (TG) mice (overexpressing superoxide dismutase and glutathione peroxidase) were randomly divided into two groups:TG+ND group and TG+HFD group. The intervention period of each group was three months. The levels of total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), non-high density lipoprotein (non-HDL) and TC/HDL in each group were determined by Alere Cholestech LDX blood lipid analyzer. The quantity changes of BMSC subsets in bone marrow and peripheral blood were analyzed by flow cytometry. Cell proliferation was detected by 5-bromo-2′-deoxyuridine labeling method. Annexin V-fluorescein isothiocyanate/propidium iodide double staining method was used to detect cell apoptosis.The level of reactive oxygen species (ROS) was detected by flow cytometric ROS detection reagent labeling method. Results The production of ROS in BMSC of HFD mice increased significantly. NAC could reverse the levels of serum TC, TG, LDL and non-LDL in HFD mice. The number of BMSC subsets Lin－c-Kit＋ cells in bone marrow of HFD mice decreased significantly, but the number of Lin－c-Kit＋ cells in blood did not decrease. The numbers of BMSC subsets Lin－Sca-1⁺ or Lin－CD133⁺ cells were similar in bone marrow and blood of HFD mice and normal diet mice. HFD significantly inhibited the proliferation and increased apoptosis of bone marrow Lin－c-Kit＋ cells in vivo. NAC treatment or transgenic mouse model effectively prevented the production of ROS in BMSC and the reduction of bone marrow Lin－c-Kit＋ cells induced by HFD. Conclusion HFD selectively decreases the number of bone marrow BMSC subsets Lin－c-Kit＋ cells by increasing the production of ROS in elderly mice.