Objective  To investigate the effects of granulocyte colony stimulating factor (G-CSF) on the expression level of miR-146a/Smad4 of leukemia cell line HL-60 and proliferative activity of leukemia cells. Methods  Human leukemia cell line HL-60 was treated 48 h in advance and controls were untreated leukemia cells. CCK8 was applied to detect the cell proliferation and flow cytometry was used to detect the changes of cell cycle. The mRNA expressions of miR-146a and Smad4 were detected by realtime quantitative PCR. The total protein expression of Smad4 was detected by Western blotting. Results  G-CSF upregulated the expression of leukemia cells miR-146a, down-regulated the mRNA expression and total protein expression of Smad4, and increased the proliferative activity of leukemia cells. Both mRNA expression and total protein expression of Smad4 of leukemia cells transfected by plasmids with overexpressed miR-146a decreased. The increase of expression of miR-146a and decrease of expression of Smad4 of transfected cells that were treated by G-CSF were more significant. Conclusion  G-CSF affects the expression of Smad4 via regulating the signal pathway of miR-146a/Smad4 and stimulates leukemia cells in quiescent state entering the proliferating phase.