Granulocyte colony-stimulating factor (G-CSF) regulates the proliferation and differentiation of neutrophilic progenitor cells. Here, we investigated the roles of CCAAT/enhancer-binding protein (C/EBP)alpha in the G-CSF-induced transcriptional activation and chromatin modification of the CCR2 and myeloperoxidase (MPO) genes in IL-3-dependent myeloid FDN1.1 cells. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays revealed that G-CSF activates C/EBPalpha to bind target promoters. ChIP mapping experiments across the CCR2 and MPO genes showed that G-CSF induces histone H3 modifications: the acetylation of Lys9, trimethylation of Lys4 and trimethylation of Lys9. The distribution profile of the trimethylated Lys9 was distinct from that of the two other modifications. All the G-CSF-induced C/EBPalpha recruitment, transcriptional activation and histone modifications were reversed by re-stimulation with IL-3, and were abolished by short hairpin RNA (shRNA)-mediated knockdown of C/EBPalpha. These results indicate that C/EBPalpha is activated by G-CSF to bind target promoters, and plays critical roles in the transcriptional activation and dynamic chromatin modification of target genes during neutrophil differentiation.