Abstract: As a novel and effective tool for gene function analysis, RNA interference (RNAi) technique has been developed rapidly and made great progress in mammalian cell studies. The mechanisms and experimental operation of RNAi in mammalian cells have become research hotspots, and RNAi mechanisms were supposed to be different from those in plant. In this paper, a review of RNAi mechanisms, the latest research progress and the experimental strategies of RNAi in mammalian cells were presented, including siRNA sequence design, siRNA delivery approaches and RNAi effect detection, so as to offer valuable references for RNAi studies.