Objective To establish a method for distinguishing microglia and infiltrating macrophages in central nervous system by fluorescence-activated cell sorting. Methods Adult C57BL/6 mice with bilateral common carotid artery stenosis were treated with PLX5622 (a colony stimulating factor 1 receptor inhibitor) or clodronate liposomes. After separating, homogenizing and resuspending brain and spinal cord tissue, the mononuclear cell suspensions were obtained by Percoll density gradient centrifugation. Anti-CD45, anti-CD11b, and anti-lymphocyte antigen 6 family member C (Ly6C) were used to obtain microglia (CD11b⁺CD45^lowLy6C^- cells) and infiltrating macrophages (CD11b⁺CD45^highLy6C⁺ cells) by fluorescence-activated cell sorting. The treatment effects of PLX5622 and clodronate liposomes were verified. Results Microglia and infiltrating macrophages in the central nervous system were effectively distinguished by anti-CD45, anti-CD11b, and anti-Ly6C. Compared with the control group, the number of microglia was significantly decreased after PLX5622 treatment (P=0.001), while the number of infiltrating macrophages was significantly decreased after clodronate liposome treatment (P<0.001). Conclusion The established method can effectively distinguish microglia and infiltrating macrophages in central nervous system by fluorescence-activated cell sorting, and PLX5622 and clodronate liposomes can selectively eliminate microglia and infiltrating macrophages in the central nervous system.