MicroRNAs (miRNAs) are small single-stranded regulatory RNAs capable of interfering with messenger RNAs (mRNAs) through complete or partial complementarities. Partial complementarity gives miRNAs a flexibility which is useful for construction of new therapies against cancer polymorphisms and viral mutations. Varieties of miRNAs have been reported in diverse species; and they are believed to induce RNA interference (RNAi), a post-transcriptional gene silencing mechanism. Recently, many intronic sequences have been shown to encode microRNAs. Intronic miRNA, a new class of miRNAs, is derived from introns by RNA splicing and Dicer processing, and it differs uniquely from previously described intergenic miRNA in that intronic miRNAs require type II RNA polymerases (Pol-II) and spliceosomal components for their biogenesis. Several kinds of intronic miRNAs have been identified; however, their functions and applications have not been reported. Here, we show for the first time that intron-derived miRNAs are able to induce RNA interference in many cells demonstrating the evolutionary preservation of this post-transcriptional regulatory system in vivo.