EZH2, a core component of polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through histone H3 Lys-27 trimethylation and is involved in various biological processes, including hematopoiesis. It is well known that 3-deazaneplanocin A (DZNep), an inhibitor of S-adenosylmethionine-dependent methyltransferase that targets the degradation of EZH2, preferentially induces apoptosis in various hematological malignancies, suggesting that EZH2 may be a new target for epigenetic treatment. Because PRC2 participates in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation, inhibition of EZH2 may influence erythropoiesis. To explore this possibility, we evaluated the impact of DZNep on erythropoiesis. DZNep treatment significantly induced erythroid differentiation of K562 cells, as assessed by benzidine staining and quantitative RT-PCR analysis for representative erythroid-related genes, including globins. When we evaluated the effects of DZNep in human primary erythroblasts derived from cord blood CD34-positive cells, the treatment significantly induced erythroid-related genes, as observed in K562 cells, suggesting that DZNep induces erythroid differentiation. Unexpectedly, siRNA-mediated EZH2 knockdown had no significant effect on the expression of erythroid-related genes. Transcriptional profiling of DZNep-treated K562 cells revealed marked up-regulation of SLC4A1 and EPB42, previously reported as representative targets of the transcriptional corepressor ETO2. In addition, DZNep treatment reduced the protein level of ETO2. These data suggest that erythroid differentiation by DZNep may not be directly related to EZH2 inhibition but may be partly associated with reduced protein level of hematopoietic corepressor ETO2. These data provide a better understanding of the mechanism of action of DZNep, which may be exploited for therapeutic applications for hematological diseases, including anemia.