RNA interference (RNAi) is a process in which small-interfering RNA (siRNA) induces a sequence-dependent degradation of mRNA. In recent years, vector-based siRNA expression systems have been developed by several groups. In these systems, short-hairpin RNAs (shRNAs) have been widely chosen for first transcriptional products which are processed to siRNAs by Dicer-dependent cleavage. In this study, we demonstrate that overexpression of Dicer enhances the effect of shRNA-dependent gene silencing in mammalian cells. Therefore, these results indicate that Dicer-dependent cleavage of shRNAs is an important step for shRNA-mediated gene silencing.