Glioblastoma multiforme is characterized by high accumulation of microglia/macrophages. The function of these tumor-infiltrating myeloid cells is not sufficiently elucidated. Therefore, a better understanding of the precise immune cell composition and function in brain tumors is required. In rodent glioma models, two different myeloid cell populations exist, determined by the expression level of CD45, namely CD11b(+)CD45(low) and CD11b(+)CD45(high). Previous analyses of cytokine and marker expression profiles were almost exclusively performed on the entire myeloid cell fraction. Consequently, described pro- and anti-tumoral characteristics were not assigned to the evident subpopulations. In the present study, we used a syngeneic glioblastoma mouse model and subsequent flow cytometric analyses to demonstrate the distinct properties of CD11b(+)CD45(high) and the CD11b(+)CD45(low) cells. First, the majority of CD11b(+)CD45(high) cells expressed high level of GR1 and around 6% of IL10 representing in part features of myeloid-derived suppressor cells, while the CD11b(+)CD45(low) fraction displayed no upregulation of these molecules. Second, we detected that specifically the CD11b(+)CD45(high) population showed antigen-presenting, co-stimulatory, and inflammatory features. Here, we identified up to 80% of MHCII and approximately 50% of CD86 and TNFα-expressing cells. Investigation of MHCI and CD80 revealed a moderate upregulation. By contrast, in the CD11b(+)CD45(low) cell fraction, merely MHCII and TNFα were marginally overexpressed. In summary, these data emphasize the specific phenotype of CD11b(+)CD45(high) cells in glioma with suppressive as well as pro-inflammatory characteristics whereas the CD11b(+)CD45(low) cells were almost unaffected. Hence, primarily, the subpopulation consisting of CD45(high)-expressing cells is activated by the tumor and should be considered as therapeutic target.