RNA interference (RNAi) is a sequence-specific gene-silencing phenomenon mediated by double-stranded RNA. RNAi is made possible through the activity of an evolutionarily conserved RNA-protein machinery that is utilized by microRNAs (miRNAs), endogenously produced 21- to 23-nucleotide noncoding RNAs. As RNA-based therapeutics come of age, understanding the miRNA pathway and the mechanism of posttranscriptional gene silencing is paramount. Studies have pointed to a critical role for miRNAs in human development and disease and revealed an unanticipated complexity in the mechanisms of regulation of gene expression. This review discusses advances in the biogenesis of miRNAs, their modes of action, and implications for RNA-based treatments.