Background: Tumor-associated macrophages (TAMs) are principal immune cells in glioma microenvironment which support tumor growth and proliferation. Our aim in this study was to assess the relationship between CD204-expressed TAMs and O6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation in World Health Organization (WHO) grade 4 astrocytomas, and its impact on patient's clinical outcome. Methods: The expression of CD204 + TAMs was quantitively assessed on 45 samples of WHO grade 4 astrocytomas using immunohistochemistry. MGMT-promoter methylation was tested by methylation techniques. The relationship between TAMs, MGMT-promoter methylation, and recurrence-free interval (RFI) was statistically analyzed. Results: There were 10 cases (22.2%) with isocitrate dehydrogenase (IDH)-mutant grade 4 astrocytoma and 35 cases (77.8%) with IDH-wildtype glioblastoma. MGMT-promotor was methylated in 18 cases (40%), unmethylated in 15 cases (33%), and the remaining 12 cases showed no MGMT status because of nucleic acid degradations. The expression of CD204+ TAMs was high in 32 cases (71.7%) and low in 13 cases (28.8%). The relationship between IDH1 mutation and CD204+ TAM expression was insignificant (P = 0.93). However, the significant difference was found between MGMT methylation and CD204+ TAMs expression (P = 0.01), in which CD204+ TAMs were diffusely expressed in MGMT-methylated cases. There was no significant difference in RFI between CD204+ TAMs expression, MGMT-promoter methylation and treatment modalities. Conclusions: Grade 4 astrocytomas with diffusely expressed CD204+ TAMs are usually associated with MGMT-promoter methylation. Although this association is unclear, CD204+ TAMs may neutralize the effect of MGMT-DNA protein to loss its function, which contributes to tumor progression. This relationship had no significant impact on the patient's clinical outcome after different treatment modalities.