Granulocyte colony-stimulating factor (G-CSF) is a common inducer of the release of hematopoietic progenitor cells (HPC) from the bone marrow into the peripheral blood. However, the molecular mechanisms underlying this action of G-CSF have not been fully elucidated. Herein, we demonstrate that G-CSF is a powerful chemotactic agent for human HPC other than modulating the adhesion molecules expressed on HPC or bone marrow stromal cells. G-CSF directly chemoattracted HPC in transwell assay and this chemotaxis is time dependent and is specifically neutralized with antibodies that target its receptor. The number of cells transmigrated through the transwell toward G-CSF stimuli was more than that of stromal cell-derived factor-1 at every concentration. G-CSF induced a rapid, transient increase in F-actin polymerization and the formation of focal contact rings in HPC, which are prerequisites for cell migration. The mechanism of G-CSF-induced chemotaxis appears to involve the phosphorylation of JAK1/STAT3 pathway. Collectively, these results provide evidence that G-CSF promotes chemotactic functionality and suggests new avenues of investigation relevant to the mobilization of HPC.