MicroRNAs (miRNAs) are a class of small regulatory RNAs that are thought to regulate the expression of as many as one-third of all human messenger RNAs (mRNAs). miRNAs are thought to be involved in diverse biological processes, including tumorigenesis. Analysis of miRNA levels may have diagnostic implications. Evidence shows that numerous viruses interact with the miRNA machinery, and that a number of viruses encode their own miRNAs. It seems likely that miRNAs will be implicated in many human diseases. Manipulation of miRNA levels by gene therapy provides an attractive new approach for therapeutic development. This review focuses on approaches to manipulate miRNA levels in cells and in vivo, and the implications for gene therapy. Furthermore, we discuss the use of endogenous miRNAs as scaffolds for the expression of RNA interference (RNAi) as well as competition between exogenous RNAi triggers and endogenous miRNAs. Because short interfering RNAs can also act as miRNAs, seed matches with the 3' untranslated regions of genes should be avoided to prevent off-target effects. Last, we discuss the use of miRNAs to avoid immune responses to viral vectors.