RNA interference (RNAi) can mediate the long- or short-term silencing of gene expression at the DNA, RNA, and/or protein level. Although several triggers of RNAi have been identified, the best characterized of these are small interfering RNAs (siRNAs), which can decrease gene expression through mRNA transcript cleavage, and endogenous microRNAs (miRNAs), which primarily inhibit protein translation. An improved understanding of RNAi has provided new, powerful tools for conducting functional studies in a gene-specific manner. In various applications, RNAi has been used to create model systems, to identify novel molecular targets, to study gene function in a genome-wide fashion, and to create new avenues for clinical therapeutics. Here, we review many of the ongoing applications of RNAi in mammalian and human systems, and discuss how advances in our knowledge of the RNAi machinery have enhanced the use of these technologies.