PU.1 is a key transcription factor for hematopoiesis and plays important roles in various hematological malignancies. To clarify the molecular function of PU.1, we initially tried to identify bona fide target genes regulated by PU.1. Dual microarrays were employed for this study to compare PU.1-knockdown K562 cells (K562PU.1KD) stably expressing PU.1 short inhibitory RNAs versus control cells and PU.1-overexpressing K562 cells (K562PU.1OE) versus control cells. In these analyses, we found that several genes, including metallothionein (MT)-1 isoforms (MT-1G and MT-1A) and vimentin (VIM), were markedly induced while Jun dimerization protein (JDP) 2 was suppressed in K562PU.1KD cells. Furthermore, the mRNA expressions of the MT-1 and VIM genes were inversely correlated and the mRNA expression of JDP2 was positively correlated with PU.1 mRNA expression in 43 primary acute myeloid leukemia specimens (MT-1G: R = -0.50, p < 0.001; MT-1A: R = -0.58, p < 0.0005; VIM: R = -0.39, p < 0.01; and JDP2: R = 0.30, p < 0.05). Next, we analyzed the regulation of the MT-1 and VIM genes. We observed increased associations of acetylated histones H3 and H4 with the promoters of these genes in K562PU.1KD cells. Sequence analyses of the regions approximately 1 kb upstream from the transcription start sites of these genes revealed numerous CpG sites, which are potential targets for DNA methylation. Chromatin immunoprecipitation assays revealed that methyl CpG-binding protein 2 (MeCP2) and PU.1 bound to the CpG-rich regions in the MT-1 and VIM promoters. Bisulfite sequencing analyses of the PU.1-bound regions of these promoters revealed that the proportions of methylated CpG sites were tightly related to the PU.1 expression levels.