Objective：To examine the ability of purified 3 Trans-Activator of Transcription-RNA dinding domain（3TAT-RBD） in the delivery of siRNA into cells and to evaluate its effect on gene silencing. Methods：The RNA binding domain（Motif1） of double-stranded RNA-activated protein kinase（PKR） was fused with cell-penetrating peptide HIV-TAT to construct 3TAT-RBD by molecular cloning technique. The recombinant 3TAT-RBD protein was expressed in BL21 bacteria and was further purified with GST purification system. The binding affinity of 3TAT-RBD with Cy3-dsRNA was examined after nondenaturing gel electrophoresis. Fluorescence microscopy was used to examine the efficiency of 3TAT-RBD and Lipofectimine 2000 in the delivery of dsRNA into gli36 cells. Moreover，the si－lencing effect of 3TAT-RBD and Lipofectimine 2000 on expression of Drp1（a protein regulating mitochondrial fission） in gli36 cells was compared. Results：3TAT-RBD protein was successfully expressed and purified. In vitro binding experiments showed that 3TAT-RBD had high binding affinity with Cy3-siRNA. Both 3TAT-RBD and Lipofectimine 2000 delivered Cy3-siRNA into gli36 cells with similar efficiency. In RNAi experiments，siRNA delivered into gli36 cells by 3TAT-RBD effectively silenced Drp1 expression，and its silencing efficiency was similar to that of Lipofectamine 2000. The expression level of Drp1 in 3TAT-RBD and Lipofectamine 2000 group was respectively 42% and 36% of control. Conclusions：Advantage of cell-penetrating peptide HIV-TAT and dsRNA binding feature of Motif1 in PKR is used and a novel tool protein 3TAT-RBD is constructed to effectively deliver siRNA into cells for gene si－lencing.