The successful establishment of rat ES cell lines enables possible genetic manipulation in rats. Using homologous recombination to modify ES cell genes provides a foundation to temporal and tissue-specific knockout rats. In this review, the author introduces the establishment process of rat ES cell, summarizes its culture and identification skills, and analyzes the weakness and strength of various rat knockout techniques. In the context of increasing research on stem cell, knockout technology based on rat ES cells, which allows the most efficient modification of specific genes, shall contribute more to elucidate gene function, dissect the genetic mechanisms of human diseases and identify potential targets for drug development.