We have been developing a unique system for the downregulation of a gene expression through cutting a specific mRNA by the long form of tRNA 3'-processing endoribonuclease (tRNase ZL) under the direction of small-guide RNA (sgRNA). However, the efficacy of this system and the involvement of tRNase ZL in the living cells were not clear. Here we show, by targeting the exogenous luciferase gene, that the efficacy of the sgRNA/tRNase ZL method can become comparable to that of the RNA interference technology and that the gene silencing is owing to tRNase ZL directed by sgRNA not owing to a simple antisense effect. We also show that tRNase ZL together with sgRNA can downregulate expression of the endogenous human genes Bcl-2 and glycogen synthase kinase-3? by degrading their mRNAs in cell culture. Furthermore, we demonstrate that a gene expression in the livers of postnatal mice can be inhibited by an only seven-nucleotide sgRNA. These data suggest that sgRNA might be utilized as therapeutic agents to treat diseases such as cancers and AIDS.