Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic stem cells characterized by ineffective hematopoiesis. The DNA hypomethylating agents 5-azacytidine (5AC) and 5-aza-2'-deoxycytidine (DAC) are effective treatments for patients with MDS, increasing time to progression to acute myelogenous leukemia and improving overall response rates. Although genome-wide increases in DNA methylation have been documented in bone marrow cells from MDS patients, the methylation signatures of specific gene promoters have not correlated with the clinical response to these therapies. Recently, attention has been drawn to the potential etiologic role of decreased expression of specific ribosomal proteins in MDS and in other bone marrow failure (BMF) states. We therefore asked whether ribosomal RNA (rRNA) expression is dysregulated in MDS. We found significantly decreased rRNA expression and increased rDNA promoter methylation in CD34+ hematopoietic progenitor cells (HPC) from the majority of MDS patients as compared to normal controls. Treatment of myeloid cell lines with DAC resulted in a significant decrease in the methylation of the rDNA promoter and an increase in rRNA levels. These observations suggest that an increase in rDNA promoter methylation can result in decreased rRNA synthesis that may contribute to defective hematopoiesis and BMF in some individuals with MDS.