In attempts to effectively improve RNAi function, we herein report a new RNAi approach using X-shaped RDNA and Dgel (RNA interfering DNA hydrogel, Ri-Dgel). X-shaped RDNA is a 4 branched nanostructure which was composed of three dsDNA branches and one dsRNA branch, and the structure was made by annealing partially complementary ssDNAs and chimeric RNA–DNA oligonucleotides. Ri-Dgel was synthesized through the ligation of the X-shaped RDNAs using their palindromic sticky ends. In MDCK/GFP cells transfected with 1 μM of each format of siRNA, Ri-Dgel and X-RDNA, the intensity of GFP fluorescence was significantly reduced by 65% and 56%, respectively, while dsRNA which is a conventional siRNA format showed a relatively weak reduction intensity of 7% compared with a negative control. We also observed the decreased intensity of GFP fluorescence by approximately 59% in MDA-MB-231/GFP cells transfected with 5 nM Ri-Dgel. Furthermore, the Ri-Dgel showed persistent RNAi efficiency up to 6 days from the treatment. The use of Ri-Dgel to trigger RNAi resulted in enhanced efficacy and longer duration at lower concentration compared to traditional dsRNA implying the nanostructured DNA–RNA hybrid materials have great potential as a platform technology for RNAi-based therapy.