With the completion of human genome sequencing, the next challenge is to understand the function of each gene. RNA interference (RNAi) library can be used in large scale loss-of-function and phenotype screening. Although RNAi libraries have been proved to be a powerful tool of functional genetic screening in model organisms such as Caenorhabditis elegans, their use in mammalian cells has been hampered by toxic effect induced by long dsRNA. However, since 2003, several RNAi libraries that can be used in mammalian cells have been established and used for functional genetic screening. As a simple, effective, large scale and high-throughput screening technique in functional genomics, RNAi library can be used in functional genetic screening, drug target discovery and validation, disease gene discovery and many other areas.