CCAAT/enhancer-binding protein alpha (C/EBPalpha) is a critical regulator for early myeloid differentiation. Mutations in C/EBPalpha occur in 10% of AML patients, leading to the expression of a 30kDa dominant negative isoform (C/EBPalphap30). In the present study, using a global proteomics approach to identify the target proteins of C/EBPalphap30, we show that Ubc9, an E2 conjugating enzyme, essential for sumoylation is increased in its expression when C/EBPalphap30 is induced. We confirmed the increased expression of Ubc9 in AML patients with C/EBPalphap30 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/EBPalphap42 decreases the transactivation capacity on a minimal C/EBPalpha promoter. Importantly, overexpression of C/EBPalphap30 in 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 Ubc9 as an important C/EBPalphap30 target through which C/EBPalphap30 enhances the sumoylation of C/EBPalphap42 to inhibit granulocytic differentiation.