Summary The effect of granulocyte colony‐stimulating factor (G‐CSF) on human neutrophil motility was studied using videomicroscopy. Stimulation of neutrophils with G‐CSF resulted in enhanced motility with morphological change and increased adherence. Enhanced neutrophil motility was detected within 3–5 min after G‐CSF stimulation, reached a maximum at 10 min, and was sustained for approximately 35 min. The maximum migration rate was 84·4 ± 2·9 μm/5 min. A study using the Boyden chamber method revealed that G‐CSF‐stimulated neutrophils exhibited random migration but not chemotaxis. Enhanced neutrophil motility and morphological change were inhibited by MEK [mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK) kinase] inhibitors (PD98059 and U0126), and a phosphatidylinositol 3‐kinase (PI3K) inhibitor (wortmannin), but not by a p38 MAPK inhibitor (SB203580). These findings are consistent with the fact that G‐CSF selectively activates MEK/ERK and PI3K, but not p38, in neutrophils. MEK/ERK activation was associated with G‐CSF‐induced redistribution of F‐actin and phosphorylated myosin light chain. Enhanced neutrophil motility was observed even in the presence of neutralizing anti‐CD18 antibody, which prevented cell adherence. These findings indicate that G‐CSF induces human neutrophil migration via activation of MEK/ERK and PI3K.