Gene silencing mechanisms that are mediated by small RNAs of 20-30 nucleotides(nt) are collectively called RNA silencing. The representative mechanism is RNA interference (RNAi), in which ~21-nt small RNAs (short interfering RNAs or siRNAs)efficiently trigger cleavage of target gene transcripts. As a result, proteins are no longer made from the targeted mRNAs. RNAi is tremendously specific and efficient with regard to recognizing target gene and disrupting their expression. The triggering molecules, double-stranded (ds) RNAs, are quite stable in vivo and are basically indistinguishable from natural endogenous RNAs; thus, RNAi intrinsically has great potential for therapeutic use. In fact, RNAi-based clinical trials for treating human diseases are currently ongoing worldwide. However, optimization of the protocols still requires substantial investigation; indeed, relevant studies have been undertaken internationally by many researchers. Given the growing anticipation of RNAi as a disease therapy, re-examining the RNAi mechanism and machinery from a biochemical perspective might provide a way to advance its successful application in the human body; this, therefore, is the focus of this Commentary.