CCAAT/enhancer-binding protein alpha (C/EBP alpha) is an essential transcription factor for myeloid lineage commitment. Here we demonstrate that acetylation of C/EBP alpha at lysine residues K298 and K302, mediated at least in part by general control non-derepressible 5 (GCN5), impairs C/EBP alpha DNA-binding ability and modulates C/EBP alpha transcriptional activity. Acetylated C/EBP alpha is enriched in human myeloid leukaemia cell lines and acute myeloid leukaemia (AML) samples, and downregulated upon granulocyte-colony stimulating factor (G-CSF)-mediated granulocytic differentiation of 32Dcl3 cells. C/EBP alpha mutants that mimic acetylation failed to induce granulocytic differentiation in C/EBP alpha-dependent assays, in both cell lines and in primary hematopoietic cells. Our data uncover GCN5 as a negative regulator of C/EBP alpha and demonstrate the importance of C/EBP alpha acetylation in myeloid differentiation.