Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into approximately 21- to 27-nt products that trigger sequence-directed gene silencing by RNA interference. Although the mechanism of RNA recognition and length-specific cleavage by Dicer has been established, the way in which dicing activity is regulated is unclear. Here, we show that the N-terminal domain of human Dicer, which is homologous to DExD/H-box helicases, substantially attenuates the rate of substrate cleavage. Deletion or mutation of this domain activates human Dicer in both single- and multiple-turnover assays. The catalytic efficiency (k(cat)/K(m)) of the deletion construct is increased by 65-fold over that exhibited by the intact enzyme. Kinetic analysis shows that this activation is almost entirely due to an enhancement in k(cat). Modest stimulation of catalysis by the full-length Dicer enzyme was observed in the presence of the TAR-RNA binding protein, which physically interacts with the DExD/H-box domain. These results suggest that the DExD/H-box domain likely disrupts the functionality of the Dicer active site until a structural rearrangement occurs, perhaps upon assembly with its molecular partners.