MicroRNAs (miRNAs) are initially expressed as long transcripts that are processed in the nucleus to yield ∼65-nucleotide (nt) RNA hairpin intermediates, termed pre-miRNAs, that are exported to the cytoplasm for additional processing to yield mature, ∼22-nt miRNAs. Here, we demonstrate that human pre-miRNA nuclear export, and miRNA function, are dependent on Exportin-5. Exportin-5 can bind pre-miRNAs specifically in vitro, but only in the presence of the Ran-GTP cofactor. Short hairpin RNAs, artificial pre-miRNA analogs used to express small interfering RNAs, also depend on Exportin-5 for nuclear export. Together, these findings define an additional cellular cofactor required for miRNA biogenesis and function.