Abstract: Objective To investigate the effect of granulocyte-macrophage colony-stimulating factor (GMCSF) combined with temozolomide (TMZ) on high-grade glioma cells and related mechanism. Methods We cultured six cases of high-grade glioma cells from patient's tumor tissues. MTT assay was used to detect cell proliferation and toxicity. Flow cytometry was used to detect cell cycle and apoptosis rate. Specific PCR and immunofluorescence were used to detect the expression of O6-methylguanine-DNA methyltransferase (MGMT) methylation status and MGMT protein respectively. Results MTT assay suggested that compared with the control group, GM-CSF group had increased cell viability in varying degrees. In three cases of cells (MGMT gene methylation), the cell viability of the combination group (67.67±1.16), (68.13±1.06), (68.42±1.73)was significantly lower than that of the corresponding TMZ group (90.00±1.73), (82.33±1.53), (82.67 ±2.11) (P<0.05). However, there was no significant difference between the two groups in another three cases (MGMT gene unmethylated) (P>0.05). For the MGMT methylation cells, the apoptosis rate in combination group was higher than that in the corresponding TMZ group(P<0.05), which coincided with MTT assay results. In all 6 cases of primary glioma cells, GM-CSF treated group showed significant reduction in the fraction of cells in G1 phase with concomitant increase in S phase(P<0.05). Conclusion GM-CSF could induce high-grade glioma cells entering the cell cycle rapidly, which could enhance the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation.However this effect is not ideal on glioma cells with MGMT unmethylation.