Although Sirt2 is primarily expressed in oligodendrocytes of the central nervous system, its role in oligodendroglial lineage differentiation is not fully understood. Our findings demonstrate that the transcription factor Nkx2.2 binds to the Sirt2 promoter via histone deacetylase 1 (HDAC-1), the binding site for Nkx2.2 maps close to the start codon of the Sirt2 gene, and Nkx2.2 negatively regulates Sirt2 expression in CG4 cells, an oligodendroglial precursor cell line. HDAC-1 knock-down not only significantly attenuates the binding capacity of Nkx2.2 to the Sirt2 promoter but also releases repression of Sirt2 expression by Nkx2.2. Nkx2.2 over-expression down-regulates Sirt2 expression and delays differentiation of CG4 cells; in contrast, up-regulation of Sirt2 does not impact Nkx2.2 expression level. Sirt2 knock-down via RNAi or inhibition of Sirt2 by sirtinol, a Sirt2 activity inhibitor, blocks CG4 cell differentiation. Over-expression of Sirt2 facilitates CG4 cell differentiation at both molecular and cellular levels, enhancing expression of myelin basic protein and facilitating the growth of cell processes. We have conclusively demonstrated that Sirt2 enhances CG4 oligodendroglial differentiation and report a novel mechanism through which Nkx2.2 represses CG4 oligodendroglial differentiation via Sirt2.