Background: Because of relatively lower chemotherapy-related side effects and better tolerance, granulocyte colony-stimulating factor (G-CSF) combining with other low dosage chemotherapeutics has been widely used in elderly acute myeloid leukemia (AML) patients in China. Unfortunately not all patients benefit from this regimen. Therefore, identifying novel treatment protocols with minimal toxicities and overcoming drug resistance remain the most challenging scenarios facing this population of patients. Arsenic trioxide (ATO) shows its effects in acute promyelocytic leukemia treatment and may show promising effects in other subtypes of acute myeloid leukemia. The aquaporin 9 (AQP9) expression on the leukemia membrane limits ATO transporting into the leukemia cells. Therefore, we hypothesize that the combination of ATO and specific factor which may regulate AQP9 expression would increase arsenic-induced cytotoxicity in AML. Aims: To evaluate the feasibility of combining G-CSF and arsenic trioxide in AML and to explore the mechanisms underlying the increased sensitivity of AML cells to ATO induced by G-CSF. Methods: Acute myeloid leukemia HL-60 and THP-1 cells were treated with G-CSF and/or ATO. AQP9 expression was suppressed by culturing these leukemia cells with phloretin. Cell viability, apoptosis and cell cycle distribution of each group were assessed by CCK-8 and flow cytometry respectively. Mechanistic studies were carried out by RT-qPCR and western blotting. Results: Compared with single-agent treatment in HL-60 and THP-1 cells, the combination of G-CSF and arsenic trioxide was associated with decreased cell proliferation and increased apoptosis as well as altered cell cycle distribution. Mechanistically, G-CSF treatment triggered up-regulation of AQP9, which was found to control arsenic transport and might determine ATO sensitivity. G-CSF also increased phosphorylation P38 expression. Furthermore, G-CSF induced AQP9 expression effects can be neutralized by phloretin. Summary/Conclusion: The pretreatment AML cells with G-CSF enhanced ATO anti-leukemic effect. This effect may relate with G-CSF-driven up-regulation of AQP9 expression and may be associated with the activation of P38 MAPK signaling pathway. Our findings suggest that synergistic effects of ATO and G-CSF may provide as a new therapeutic approach for elderly AML patients.