Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with "undruggable" therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver silencing shRNA to target cells. This new approach, termed TransKingdom RNAi (tkRNAi), has several key advantages. First, tkRNAi may provide a viable means to accomplish therapeutic RNAi since non-pathogenic bacteria have a proven safety record in clinical applications. Second, tkRNAi eliminates the cost of siRNA manufacture since silencing shRNA are produced inside bacteria. Moreover, the intracellular mechanism of shRNA release inherent to tkRNAi may circumvent, or mitigate, the activation of host immune responses. Finally, tkRNAi may facilitate high-throughput in vivo functional genomics screening since bacteria-based RNAi libraries can be easily constructed, stored, reproduced and amplified, thereby allowing for the creation of a stable gene silencing system.