Down syndrome (DS), or Trisomy 21, is the most common genetic cause of cognitive impairment and congenital heart defects in the human population. Bioinformatic annotation has established that human chromosome 21 (Hsa21) harbors five microRNA (miRNAs) genes: miR-99a, let-7c, miR-125b-2, miR-155, and miR-802. Our laboratory recently demonstrated that Hsa21-derived miRNAs are overexpressed in DS brain and heart specimens. The aim of this study was to identify important Hsa21-derived miRNA/mRNA target pairs that may play a role, in part, in mediating the DS phenotype. We demonstrate by luciferase/target mRNA 3-untranslated region reporter assays, and gain-and lossof-function experiments that miR-155 and -802 can regulate the expression of the predicted mRNA target, the methyl-CpG-binding protein (MeCP2). We also demonstrate that MeCP2 is underexpressed in DS brain specimens isolated from either humans or mice. We further demonstrate that, as a consequence of attenuated MeCP2 expression, transcriptionally activated and silenced MeCP2 target genes, CREB1/Creb1 and MEF2C/Mef2c, are also aberrantly expressed in these DS brain specimens. Finally, in vivo silencing of endogenous miR-155 or -802, by antagomir intraventricular injection, resulted in the normalization of MeCP2 and MeCP2 target gene expression. Taken together, these results suggest that improper repression of MeCP2, secondary to trisomic overexpression of Hsa21-derived miRNAs, may contribute, in part, to the abnormalities in the neurochemistry observed in the brains of DS individuals. Finally these results suggest that selective inactivation of Hsa21-derived miRNAs may provide a novel therapeutic tool in the treatment of DS.The presence of three copies of all, or part, of human chromosome 21 (Hsa21) 2 results in the constellation of physiologic traits known as Down syndrome (DS) or Trisomy 21 (1).With an incidence of approximately one in 750 live births, DS is the most frequently survivable congenital chromosomal abnormality (2, 3). The phenotypes of DS are complex and variable; they include cognitive impairment, congenital heart defects, craniofacial abnormalities, gastrointestinal anomalies, leukemia, and Alzheimer disease (1-3). Experimental studies utilizing tissues derived from individuals with DS have confirmed that expression of trisomic genes is increased by ϳ50% (i.e. consistent with gene dosage) (4 -6). Recent bioinformatic annotation has established that Hsa21 harbors more than 500 genes (7, 8), including five miRNA genes (miR-99a, let-7c, miR125b-2, miR-155, and miR-802). miRNAs are a family of small, ϳ21-nucleotide long, nonprotein-coding RNAs that have emerged as key post-transcriptional regulators of gene expression (9 -11). miRNAs are processed from precursor molecules (pri-miRNAs), which are either transcribed from independent miRNA genes or are portions of introns of protein-coding RNA polymerase II transcripts. Following their processing, miRNAs are assembled into ribonucleoprotein complexes called microribonucleoproteins (miRNPs) or miRNA-...