The discovery that small interfering RNA duplexes (siRNA) can silence gene expression in mammalian cells has revolutionized biomedical research. The most successful application of the discovery has been to study gene function in cultured human or mouse cells. However, the knockdown effect of siRNA is only transient. To achieve a more sustained gene-silencing effect, shRNA (small hairpin RNA) expressed from a vector is preferred. An additional benefit of shRNA is that RNA interference (RNAi) can now be applied in vivo through delivering shRNA-expressing vectors by transgenic technology. Transgenic RNAi not only allows the study of biological processes not present in cultured cells but also offers chronic therapeutic potentials. In this review, we will summarize the developments in the generation of transgenic RNAi mice.