Aberrant DNA methylation is the most frequent molecular alteration in acute myeloid leukemia (AML). To identify methylation-silenced genes in AML, we performed microarray analyses in U937 cells exposed to the demethylating agent 5-aza-deoxy-cytidine. Overall, 274 transcripts were significantly induced. Interestingly, C/EBPδ expression was significantly induced (more than 10-fold) by demethylation whereas expression of all other C/EBP family members remained unchanged. The C/EBPδ promoter was strongly methylated in different leukemic cell lines and showed signs of a repressed chromatin state. Analyses of the promoter regions of the entire C/EBP family (α, β, γ, δ, ϵ, ζ) in bone marrow samples from AML patients (n = 80) and controls (n = 15) by mass spectrometry revealed that C/EBPδ is the most commonly hypermethylated C/EBP gene in AML. Hypermethylation occurred in more than 35% of AML patients at primary diagnosis. A significant correlation (P = .016) was observed between hypermethylation of the C/EBPδ promoter and low expression of C/EBPδ in AML patients. C/EBPδ promoter activity was strongly repressed by methylation in vitro, and transcriptional repression partially depended on MeCP2 activity. C/EBPδ exhibited growth-inhibitory properties in primary progenitor cells as well as in Flt3-ITD–transformed cells. Taken together, C/EBPδ is a novel tumor suppressor gene in AML that is silenced by promoter methylation.