Down syndrome (DS) or Trisomy 21 (Ts21) is caused by the presence of an extra copy of all or part of human chromosome 21 (Hsa21) and is the most frequent survivable congenital chromosomal abnormality. Bioinformatic annotation has established that Hsa21 harbors more than 400 genes, including five microRNA (miRNA) genes (miR-99a, let-7c, miR-125b-2, miR-155, and miR-802). MiRNAs are endogenous, single-stranded, small non-coding RNA molecules that regulate gene expression by interacting with specific recognition elements harbored within the 3'-untranslated region (3'-UTR) of mRNAs and subsequently target these mRNAs for translational repression or destabilization. MiRNA expression profiling, miRNA RT-PCR, and miRNA in situ hybridization experiments have demonstrated that Hsa21-derived miRNAs were over-expressed in fetal brain and heart specimens isolated from individuals with DS. We now propose that Ts21 gene dosage over-expression of Hsa21-derived miRNAs in DS individuals result in the decreased expression of specific target proteins (i.e. haploinsufficiency) that contribute, in part, to the DS phenotype.