AbstractCCAAT/enhancer-binding protein α (C/EBPα) is a critical regulator for early myeloid differentiation. Mutations in C/EBPα occur in 10% of patients with acute myeloid leukemia (AML), leading to the expression of a 30-kDa dominant-negative isoform (C/EBPαp30). In the present study, using a global proteomics approach to identify the target proteins of C/EBPαp30, we show that Ubc9, an E2-conjugating enzyme essential for sumoylation, is increased in its expression when C/EBPαp30 is induced. We confirmed the increased expression of Ubc9 in patients with AML with C/EBPαp30 mutations compared with other subtypes. We further confirmed that the increase of Ubc9 expression was mediated through increased transcription. Furthermore, we show that Ubc9-mediated enhanced sumoylation of C/EBPαp42 decreases the transactivation capacity on a minimal C/EBPα promoter. Importantly, overexpression of C/EBPαp30 in granulocyte colony-stimulating factor (G-CSF)–stimulated human CD34+ cells leads to a differentiation block, which was overcome by the siRNA-mediated silencing of Ubc9. In summary, our data indicate that Ubc9 is an important C/EBPαp30 target through which C/EBPαp30 enhances the sumoylation of C/EBPαp42 to inhibit granulocytic differentiation.