MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a post-transcriptional gene silencing mechanism. Intronic miRNAs, derived from introns by RNA splicing and Dicer processing, can interfere with intracellular mRNAs to silence that gene expression. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of type II RNA polymerases (Pol-II) and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in Caenorhabditis elegans, mouse and human cells; however, neither their function nor application has been reported. To this day, the computer searching program for miRNA seldom include the intronic portion of protein-coding RNAs. The functional significance of artificially generated intronic miRNAs has been successfully ascertained in several biological systems such as zebrafishes, chicken embryos and adult mice, indicating the evolutionary preservation of this gene regulation system in vivo. Multiple miRNAs can be generated from the same cluster of introns; however, non-homologous miRNAs may have different targets and functions while homologous miRNA may be derived from different intronic clusters. Taken together, the model of intronic miRNA-mediated transgenic animals provides a tool to investigate the mechanism of miRNA-associated diseases in vivo and will shed light on miRNA-related therapies.