Glioma is the most malignant brain cancer. The neurons, macrophages, T cells and other immune cells constitute the glioma immunosuppressive microenvironment. The accurate spatial distribution of these cells in the glioma microenvironment and its relationship with glioma metastasis is unknown. We constructed a mouse glioma cell line stably expressing the large Stokes-shifted yellow fluorescent protein and applied it to the multicolor immunofluorescence imaging. The imaging data revealed that the neurons were sparsely distributed in the glioma core and the number of neurons decreased by 90% compared with normal brain site. The spatial distribution of monocyte-macrophages and microglia is heterogeneous. The monocyte-macrophages and T cells were heavily recruited into the glioma core and metastasis. There was no significant difference in the distribution of microglia among glioma core, margin, and normal brain site. Our results provided new perspectives for targeting immune regulation cells and developing new immunotherapy strategies for glioma.Glioma is the most malignant brain cancer. The neurons, macrophages, T cells and other immune cells constitute the glioma immunosuppressive microenvironment. The accurate spatial distribution of these cells in the glioma microenvironment and its relationship with glioma metastasis is unknown. We constructed a mouse glioma cell line stably expressing the large Stokes-shifted yellow fluorescent protein and applied it to the multicolor immunofluorescence imaging. The imaging data revealed that the neurons were sparsely distributed in the glioma core and the number of neurons decreased by 90% compared with normal brain site. The spatial distribution of monocyte-macrophages and microglia is heterogeneous. The monocyte-macrophages and T cells were heavily recruited into the glioma core and metastasis. There was no significant difference in the distribution of microglia among glioma core, margin, and normal brain site. Our results provided new perspectives for targeting immune regulation cells and developing new immunotherapy strategies for glioma.